WO2015091897A1 - Determination of platelet-mirnas - Google Patents
Determination of platelet-mirnas Download PDFInfo
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- WO2015091897A1 WO2015091897A1 PCT/EP2014/078633 EP2014078633W WO2015091897A1 WO 2015091897 A1 WO2015091897 A1 WO 2015091897A1 EP 2014078633 W EP2014078633 W EP 2014078633W WO 2015091897 A1 WO2015091897 A1 WO 2015091897A1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Definitions
- the present invention relates to methods, use and kits for the determination of platelet-miRNAs
- miRNAs are a new class of biomarkers. They represent a group of small noncoding RNAs that regulate gene expression at the posttranslational level by degrading or blocking translation of messenger RNA (mRNA) targets. So far, miRNAs have been extensively studied in tissue material where it was found that miRNAs are expressed in a highly tissue-specific manner. Since recently it is known that miRNAs are not only present in tissues but also in body fluid samples, including blood. Nevertheless, the mechanism why miRNAs are found in blood, especially in the cellular blood fraction (blood cells of subfractions thereof) or in the extra-cellular fraction (serum/plasma), or their function in these blood fractions is not understood yet.
- the inventors of the present invention assessed for the first time the expression of platelet- miRNAs from whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells, hence in front of a background of white blood cells and/or red blood cells. They surprisingly found that especially the platelet-miRNAs are significantly dysregulated in whole blood samples collected in whole blood collection tubes, preferably collected in Paxgene-like tubes, more preferably collected in PAXgeneTM Blood RNA tubes. This is surprising since the RNA-content of the anucleated platelets is approximately 10.000 times lower when compared to the RNA-content of the nucleated white blood cells, with which the platelets are mixed in the whole blood sample collected in said whole blood collection tubes.
- the expression of the platelet-miRNAs can be determined directly from total RNA isolated from the mixture of platelets, white blood cells and red blood cells present in the whole blood collected in said whole blood collection tubes without the need for prior depletion or removal of the white blood cells and/or the red blood cells.
- the expression of the platelet- miRNAs can be determined in front of a background of white blood cells and/or red blood cells, without the need to isolate (or enrich) the platelets or the platelet fraction from the whole blood sample before. This is of advantage to applications, especially diagnostic applications, where the determination of said platelet-miRNAs can be directly determined from a whole blood sample without the need for additional sample processing steps (e.g.
- platelet-miR As that are determined from total R A isolated from whole blood samples without prior depletion and/or removal of white blood cells and/or red blood cells (or in front of a background of white blood cells and/or red blood cells; or without prior isolation of the platelet fraction), may be employed as biomarkers for non-invasive diagnosis of diseases, platelet-related diseases or platelet-related (platelet-activated) inflammatory components of such diseases.
- platelet-miRNAs that are determined from total RNA isolated from whole blood samples without prior depletion and/or removal of white blood cells and/or red blood cells (or in front of a background of white blood cells and/or red blood cells; or without prior isolation of the platelet fraction) may be employed as biomarkers for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy.
- the inventors of the present invention found that the whole blood collection tubes, preferably the Paxgene-like tubes, more preferably the PAXgeneTM Blood RNA tubes, are suitable or useful for determination of expression profiles of platelet-miRNAs and that such expression profiles may be used in a method for non-invasive diagnosis of diseases, for non-invasive diagnosis of platelet-related diseases or for non-invasive diagnosis of platelet-related (platelet-activated) inflammatory components of such diseases and for determining platelet-activity or for monitoring the efficacy of anti-platelet therapy.
- the invention provides for a method for diagnosing a platelet-related or a platelet-activated component of a disease by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the invention provides for a method for diagnosing a disease by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the invention provides for a method for diagnosing a platelet-related disease in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the invention provides for a method for monitoring the progression of a platelet-related disease in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the invention provides for a method for determining the platelet activity in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the invention provides for a method for monitoring the efficacy of an antiplatelet therapy in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the invention provides for a method for determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the invention provides for the use of a whole blood collection tube in the method according to any of the first, second, third, fourth, fifth, sixth or seventh aspect of the invention, wherein the expression profile of a set of at least one platelet-miRNA is determined from the total RNA isolated from the whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the invention provides for a kit for use in the method in the method according to any of the first, second, third, fourth, fifth, sixth or seventh aspect of the invention, comprising:
- the expression profile and the at least one reference expression profile are obtained from said at least one platelet-miRNA selected from the group consisting SEQ ID NO : 1 to 56 and wherein the expression profile and the at least one reference expression profile are determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- test compounds As used in this specification and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents, unless the content clearly dictates otherwise. For example, the term “a test compound” also includes “test compounds”.
- micro RNA refers to single-stranded RNA molecules of at least 10 nucleotides and of not more than 35 nucleotides covalently linked together.
- the miRNAs regulate gene expression and are encoded by genes from whose DNA they are transcribed, but miRNAs are not translated into protein (i.e. miRNAs are non-coding RNAs).
- miRNAs are non-coding RNAs.
- micro RNA* refers to miRNA molecules derived from the passenger strand upon processing. In the context of the present invention, the terms “miRNA” and “miRNA*” are interchangeable used.
- the miRBase (www.mirbase.org) is a well established repository and searchable database of published miRNA sequences and annotation.
- a human miRNA may also be suitable for detecting the respective miRNA orthologue(s) in another species, e.g. in another mammal, e.g. in an animal such as mouse or rat or vice versa.
- platelet refers to the smallest type of blood cells, also known as "thrombocytes", which are released into the blood stream from bone marrow megakaryocytes. Platelets play a crucial role in haemostasis and thrombosis. Platelets do not contain a nucleus, therefore missing substantial parts of the microRNA machinery components required for transcription and nuclear processing. Platelets comprise approximately 10.000 times less RNA when compared to nucleated cells (e.g. white blood cells). Platelets contain miRNAs (PMID 21415270, 22371016, 23323973), but it is not yet completely understood where these originate from and how these interact in translational control or repression of platelet mRNAs.
- miRNAs PMID 21415270, 22371016, 23323973
- platelet-miRNA refers to a miRNA that is expressed in platelets (thrombocytes). This does not necessarily mean that this platelet- miRNA is exclusively expressed in platelets and not in any other blood cells, in other cells, in other body fluids or in tissue. Currently, there are approximately 400 miRNAs described to be expressed in platelets.
- the platelet-miRNAs according to the present invention are selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 56 and are listed in Figure 1.
- the expression level of said platelet-miRNAs selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO : 56 are in a range that allows for direct determination from a whole blood sample without the need for additional sample processing steps (e.g. removal or depletion of the red blood cells and/or white blood cell fractions, or isolation of the platelet fraction), which is of advantage to commercial applications, especially in the field of diagnostics.
- the platelet-miRNAs selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 56 can be determined directly from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the inventors of the present invention applied further criteria based on expression data in a plurality of diseases for selection of said 56 platelet-miRNAs with SEQ ID NO: 1 to SEQ ID NO: 56 from the approximately 400 miRNAs that are described to be expressed in platelets.
- the inventors investigated a plurality of diseases, including Cancer (e.g. Renal Cancer, Colon Cancer, Lung Cancer, Melanoma, Ovarian Cancer, Wilm's Tumor, Prostate Cancer), Autoimmune Diseases (e.g.
- the term "whole blood sample”, as used in the context of the present invention, refers to a blood sample originating from a subject containing all blood fractions, including both the cellular (red blood cells, white blood cells, platelets) and the extra-cellar blood fraction (serum, plasma).
- the "whole blood sample” may be derived by removing blood from a subject by conventional blood collecting techniques, but may also be provided by using previously isolated and/or stored blood samples.
- the whole blood sample from a subject e.g. human or animal
- whole blood collection tube relates to blood collection tube, that is used for collection of whole blood samples, preferebly a whole blood collection tube according to the present invention is a Paxgene-like tube, a PAXgeneTM Blood RNA tube, a Tempus Blood RNA tube, an EDTA-tube, a Na-citrate tube or a ACD-tube (Acid citrate dextrose).
- a whole blood collection tube is a Paxgene-like tube, a PAXgeneTM Blood RNA tube, a Tempus Blood RNA tube, an EDTA-tube, a Na-citrate tube or a ACD-tube (Acid citrate dextrose).
- the RNA-fraction, -especially the miRNA fraction- may be protected/guarded against degradation.
- special whole blood collection tubes e.g.
- RNAlaterTM Blood RNA tubes from Preanalytix, TempusTM Blood RNA tubes from Applied Biosystems
- additives e.g. RNAlaterTM from Ambion, RNAsin ® from Promega, RNA Retain ® from Asuragen, catrimox-1 4 o r tetradecyltrimethyl-ammonium oxalate or derivatives thereof
- RNAlaterTM from Ambion
- RNAsin ® from Promega
- RNA Retain ® from Asuragen
- Paxgene-like tube as used in the context of the present invention relates to blood collection tubes, which are suited or used for collection of whole blood samples, which contain additives for stabilization of RNA and which are suited for expression analyses of the intracellular RNA and/or intracellular miRNAs and/or platelet-miRNAs.
- additives for stabilization of RNA are employed, including, but are not limited to, RNAlaterTM, RNAsin ® , RNA Retain ® catrimox-14 or tetradecyltrimethylammonium oxalate or derivatives thereof.
- Paxgene-like tubes are “PAXgeneTM Blood RNA tubes", which are suited for collection of whole blood samples and which contain additives for cell lysis and stabilization of intracellular RNA and which are suited for expression analyses of RNAs and/or miRNAs and/or platelet-miRNAs.
- additives for cell lysis and stabilization of intracellular RNA include, but are not limited to, catrimox-1 4 or tetradecyltrimethylammonium oxalate or derivatives thereof.
- PAXgeneTM Blood RNA tubes are currently marketed by PreAnalytix ( w w w . p re a n a I yt i x .
- total RNA refers to the RNA isolated from a whole blood sample comprising the miRNA- fraction.
- the total RNA comprising the miRNA- fraction or comprising a miRNA-enriched fraction, is obtained by lysis (e.g. Trizol) of the blood cells of the whole blood sample, followed by RNA isolation (extraction) e.g.
- kits for RNA isolation and purification include the miRNeasy Kits (Qiagen), PAXgeneTM Blood miRNA Kit (Qiagen), mirVana PARIS Kit (Life Technologies), PARIS Kit (Life Technologies), Tempus Spin RNA Isolation Kit (Life Technologies).
- the total RNA according to the present invention contains the miRNA- fraction or contains a miRNA- enriched fraction.
- intracellular RNA as used herein relates to the RNA present in the blood cells (platelets, white blood cells, red blood cells) of a whole blood sample, comprising the miRNA- fractions.
- expression profile represents the a measure that correlates with the miRNA expression (level) in a sample.
- each miRNA is represented by a numerical value. The higher the value of an individual miRNA, the higher is the expression level of said miRNA, or the lower the value of an individual miRNA, the lower is the expression level of said miRNA.
- the expression profile may be generated by any convenient means, e.g. nucleic acid hybridization (e.g. to a microarray), nucleic acid amplification (PCR, RT-PCR, qRT-PCR, high-throughput RT-PCR), ELISA for quantitation, next generation sequencing (e.g.
- the sample material measured by the aforementioned means are derived from a whole blood sample (without prior depletion or removal of the white blood cells and/or the red blood cells or without isolation of the platelet fraction or in front of a background of white blood cells and/or red blood cells), comprising said platelet-miRNAs and may be total RNA, labeled total RNA, amplified total RNA, cDNA, labeled cDNA, amplified cDNA, miRNA, labeled miRNA, amplified miRNA or any derivatives that may be generated from the aforementioned RNA/DNA species.
- the "expression profile”, as used herein, relates to a collection of expression (levels) of at least one miRNAs, preferably of least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 51 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 ,39 ,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or 56 or more miRNAs.
- determining an expression profile relates to the determination of the expression profile of set comprising at least one platelet-miRNA, hence from miRNA(s) expressed in platelets, preferably from miRNAs expressed in platelets selected from the group consisting of SEQ ID NO 1 to 56.
- the determination of the expression profile is a measure that directly or indirectly correlates with the levels of platelet-miRNAs present in said whole blood sample.
- nucleic acid hybridisation nucleic acid amplification, polymerase extension, mass spectroscopy, flow cytometry, sequencing
- miRN A- isolation RNA- or miRNA-enrichment
- RNA- or miRNA-purification RNA- or miRNA-labeling
- polymerase extension of RNA or miRNA ligation of RNA or miRNA, reverse-transcription of RNA or miRNA into cDNA, amplification of the cDNA, labelling of cDNA.
- nucleic acid hybridization relates to a means for determining an expression profile.
- the nucleic acid hybridization may be performed using a microarray/biochip or in situ hybridization.
- the polynucleotides (probes) with complementarity to the corresponding platelet-miRNAs to be detected are e.g. attached to a solid phase to generate a microarray/biochip.
- Said microarray/biochip is then incubated with a sample containing the platelet-miRNA(s) (or a species that is derived from said platelet- miRNA(s)), which may be labelled or unlabelled.
- Quantification of the expression level of the miRNAs may then be carried out e.g. by direct read out of said label or by additional manipulations, e.g. by use of an enzymatic reaction.
- the polynucleotides which are at least partially complementary to miRNAs having SEQ ID NO: 1 to 56 are contacted with said sample containing said platelet-miRNA(s) (or a species that is derived from said platelet-miRNA(s), e.g. a cDNA-species) in solution to hybridize.
- the hybridized duplexes are pulled down to the surface and successfully captured miRNAs are quantitatively determined (e.g. FlexmiR-assay, FlexmiR v2 detection assays from Luminex, Fireplex assay from Firefly Bioworks).
- nucleic acid amplification relates to a means for determining an expression profile.
- Nucleic acid amplification may be performed using real time polymerase chain reaction (RT-PCR) such as real time quantitative polymerase chain reaction (RT qPCR).
- RT-PCR real time polymerase chain reaction
- the standard real time polymerase chain reaction (RT-PCR) is preferred for the analysis of a single miRNA or a set comprising a low number of miRNAs (e.g. a set of at least 2 to 10 miRNAs), whereas high-throughput RT-PCR technologies (e.g.
- OpenArray from Applied Biosystems, SmartPCR from Wafergen, Biomark System from Fluidigm are also able to measure large sets (e.g a set of 5, 10, 20, 30, 50, 80, 100, 200 or more) of miRNAs in a high parallel fashion.
- RT-PCR real time polymerase chain reaction
- steps may include the following steps: (i) extracting the total RNA from a whole blood sample collected in a whole blood collection tube, preferably collected in a Paxgene-like tube, particularly preferably collected in a PAXgeneTM Blood RNA tube, obtained from a subject, (ii) obtaining cDNA samples by RNA reverse transcription (RT) reaction using universal or miRNA-specific primers; (iii) optionally pre-amplifying the cDNA of step (ii) via polymerase chain reaction (PCR), (iv) amplifying the optionally pre-amplified cDNA via polymerase chain reaction (PCR), thereby monitoring the amplification through a previously added fluorescent reporter dye (e.g.
- Step (i) the isolation and/or extraction of RNA may be omitted in cases where the RT-PCR is conducted directly from the miRNA-containing sample.
- Kits for determining a miRNA expression profile by real time polymerase chain reaction are e.g. from Life Technologies, Applied Biosystems, Ambion, Roche, Qiagen, Invitrogen, SABiosciences, Exiqon.
- sequencing relates to a means for determining an expression profile, including conventional (Maxam-Gilbert, Sanger) sequencing technology, Pyrosequencing or next generation sequencing technology (e.g. ABI SOLID, Illumina Hiseq, Gnubio, Pacific Biosystems, Roche 454) or any other sequencing technology, capable of determination of the expression profile of set comprising at least one platelet-miRNA.
- both the expression profile of the subject to be tested as well as the reference are determined from the same platelet-miRNAs and the same sample type (collected and worked up in the same way), preferably they are determined from a whole blood sample collected in whole blood collection tubes, preferably collected in Paxgene-like tubes, more preferably collected in PAXgeneTM Blood RNA tubes.
- the reference may be a reference expression profile obtained from determining one or more expression profiles of set comprising at least one platelet-miRNA from one or more whole blood samples collected in whole blood collection tubes, preferably determined from one or more whole blood samples collected in Paxgene-like tubes, more preferably determined from one or more whole blood samples collected in a PAXgeneTM Blood RNA tubes, in one or more reference subjects.
- the reference may be an algorithm, a mathematical function or a score that was developed from such an aforementioned reference expression profile.
- diagnosis refers to the process of determining a possible disease or disorder or a certain component of such a disease (e.g.
- the diagnosis comprises (i) determining the occurrence/presence of the disease ( or of a component of a disease), especially in an (very) early phase of the disease (ii) monitoring the course or progression of the disease, (iii) staging of the disease, (iv) measuring the response of a patient affected with the disease to therapeutic intervention, (v) monitoring the efficacy of a therapeutic intervention and/or (vi) segmentation of a subject suffering from the disease.
- platelet-related component of a disease refers to an aspect or feature of the disease that is platelet-related or related to the function/dis function of platelets in said disease.
- platelet-related components include, but are not limited to, haemostasis-related, thrombosis-related, immunity-related, or inflammation-related aspects or features of the disease, more preferably the platelet-related component of a disease is an inflammatory component of the disease.
- platelet-activated component of a disease refers to an aspect or feature of the disease that is platelet-activated or activated due to function/dis function of platelets in said disease.
- platelet-activated components include, but are not limited to, haemostasis-activated, thrombosis-activated, immunity-activated, or inflammation-activated aspects or features of the disease, more preferably the platelet- activated component of a disease is an inflammatory component of the disease.
- Step 1 Providing a whole blood sample of a subject : Whole blood is drawn from a subject into a PAXgeneTM Blood RNA tube (or into another whole blood collection tube, preferably into a Paxgene-like tube), drawing of 2.5 ml of whole blood into a PAXgeneTM Blood RNA tube is sufficient for the downstream analyses. The tube should be carefully inverted to ensure that the reagents contained are thoroughly
- Step 2 Isolation of the total RNA from said whole blood sample :
- the PAXgeneTM Blood RNA tube which was allowed to incubate at room temperature for at least 1 hour, is centrifuged to form a blood cell pellet (comprising white blood cells, red blood cells, platelets) at the bottom of the tube, which is collected, whereas the supernatant is discarded.
- the total RNA (comprising the miRNA fraction) is isolated from the collected blood cell pellet (comprising white blood cells, red blood cells, platelets) using suitable kits (e.g. miRNeasy kit) and/or purification methods.
- Step 3 Determining an expression profile of a set comprising at least one platelet-miRNA from the total RNA isolated : From the total RNA isolated the expression profile of at least one platelet-miRNA, selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:
- NO: 56 is measured using experimental techniques. These techniques include but are not limited to nucleic acid hybridisation based approaches, nucleic acid amplification methods (PCR, RT-PCR, qPCR), sequencing, next generation sequencing, flow cytometry and/or mass spectroscopy. In a preferred embodiment, it may be required in Step 3 that the total RNA or that individual platelet-miRNAs are reverse-transcribed into cDNA and optionally be amplified before the expression profile is determined.
- Step 4 Comparing said expression profile to a reference : The expression profile obtained in Step 3) is compared to a reference.
- the reference may be e.g. a reference expression profile, obtained from determining one or more expression profile of said at least one platelet-miRNA from whole blood collected in the same type of whole blood collection tube - exemplarily here collected in a PAXgeneTM Blood RNA tube- as in Step 1 (or collected in a another whole blood collection tube, preferably collected into a Paxgene- like tube as in Step 1) in one or more reference subjects or the reference may be an algorithm, a mathematical function or a score that was developed from such a reference expression profile.
- Step 5 Identifying if said subject is affected by one of the disease conditions : The comparison to the reference then allows to identify if said subject is affected or not affected by the disease (and/or the platelet- related (or platelet-activated) inflammatory component of the disease and/or the platelet-related disease) or alternatively if certain thresholds for monitoring the progression of a platelet-related (platelet-activated) disease and/or if thresholds for determining the platelet activity and/or if thresholds for monitoring the efficacy of an anti-platelet therapy are reached.
- Step 6 Optionally administering said affected subject to therapy of said condition :
- said subject that was identified to be affected by the disease (and/or the platelet- related (or platelet-activated) inflammatory component of the disease and/or the platelet-related disease), is administered to therapy, e.g. by treating the subject with drugs suited for therapy of said disease (and/or drugs suited for said the platelet- related (or platelet- activated) or said inflammatory component of the disease and/or drugs suited for said platelet-related disease).
- the present invention relates to method for diagnosing a platelet-related (or platelet-activated) component of a disease by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to 56.
- the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a Paxgene-like tube, particularly preferred it is collected in a PAXgeneTM Blood RNA tube.
- the platelet-related (or platelet-activated) component of a disease is a inflammatory component of said disease.
- the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the expression profile is determined from a whole blood sample in front of a background of white blood cells and red blood cells.
- the expression profile is determined from a whole blood sample without prior isolation (or extraction) of the platelet fraction
- the method of the first aspect of the present invention comprises the following steps :
- the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2, more preferably said at least one miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 32, 34, 37, 38, 40, 42, 43, 44, 46, 47, 49, 51, 52.
- the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 14, 15, 17, 18, 21, 22, 24, 25, 27, 32, 35.
- the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 6, 7, 8, 10, 11, 15, 17, 18, 19, 23.
- the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 7, 8, 9, 1 1, 13, 16, 20, 21, 22, 25, 26, 27, 28, 30, 31, 36, 41, 45.
- the disease is Multiple Sclerosis and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 6, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 5, 1 1, 12, 13, 16, 19, 20, 21, 23, 24, 25, 26, 28, 29, 31, 32, 37, 39, 50, 55.
- the disease is Benign Prostate Hyperblasia (BPH) and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 7, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 16, 17, 18, 19, 21, 37, 40, 41.
- BPH Benign Prostate Hyperblasia
- the disease is Pancreatitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 8, more preferably said at least one miRNA is selected from the group SEQ ID NO: 12, 23, 26, 27, 29, 33, 35.
- the disease is Wilms' Tumor and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 9, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 30, 41, 46, 53.
- the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 9, 1 1, 12, 14, 19, 22, 26.
- the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 11, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 22, 23, 35, 39, 56.
- the disease is Pancreatic Cancer and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 12, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 12, 27, 29, 33.
- the disease is Periodontitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 13, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 9, 10, 1 1, 12, 14, 15, 16, 20, 21, 23, 24, 25, 29, 30, 33, 34, 39.
- the disease is COPD and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 14, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 10, 13, 14, 16, 17, 18, 26, 36, 42, 43, 48.
- the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 5, 8, 9, 13, 14, 19, 22, 28, 33, 34, 38, 54.
- the disease is Ovarian Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 16, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 5, 6, 1 1 , 13, 16, 17, 19, 21 , 22, 23, 24, 27, 28, 30, 39, 42, 44, 46, 49.
- the set comprising at least one platelet-miRNA does not comprise miRNA(s) selected from the group consisting of hsa-miR-126, hsa-miR-197, hsa-miR-223, hsa-miR-21 and hsa-miR-24.
- the invention in a second aspect, relates to a method for diagnosing a disease by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to 56.
- the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a Paxgene-like tube, particularly preferred it is collected in a PAXgeneTM Blood RNA tube.
- the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the expression profile is determined from a whole blood sample in front of a background of white blood cells and red blood cells.
- the expression profile is determined from a whole blood sample without prior isolation (or extraction) of the platelet fraction
- the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2, more preferably said at least one miRNA is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 32, 34, 37, 38, 40, 42, 43, 44, 46, 47, 49, 51, 52.
- the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 14, 15, 17, 18, 21, 22, 24, 25, 27, 32, 35.
- the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 6, 7, 8, 10, 11, 15, 17, 18, 19, 23.
- the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 11 , 13, 16, 20, 21 , 22, 25, 26, 27, 28, 30, 31, 36, 41, 45.
- the disease is Multiple Sclerosis and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 6, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 5, 11 , 12, 13, 16, 19, 20, 21 , 23, 24, 25, 26, 28, 29, 31, 32, 37, 39, 50, 55.
- the disease is Benign Prostate Hyperblasia (BPH) and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 7, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 16, 17, 18, 19, 21, 37, 40, 41.
- BPH Benign Prostate Hyperblasia
- the disease is Pancreatitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 8, more preferably said at least one miRNA is selected from the group SEQ ID NO: 12, 23, 26, 27, 29, 33, 35.
- the disease is Wilms' Tumor and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 9, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 30, 41, 46, 53.
- the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 9, 11, 12, 14, 19, 22, 26.
- the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 11 , more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 22, 23, 35, 39, 56.
- the disease is Pancreatic Cancer and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 12, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 12, 27, 29, 33.
- the disease is Periodontitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 13, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 9, 10, 1 1 , 12, 14, 15, 16, 20, 21, 23, 24, 25, 29, 30, 33, 34, 39.
- the disease is COPD and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 14, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 10, 13, 14, 16, 17, 18, 26, 36, 42, 43, 48.
- the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 5, 8, 9, 13, 14, 19, 22, 28, 33, 34, 38, 54.
- the disease is Ovarian Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 16, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 5, 6, 1 1 , 13, 16, 17, 19, 21, 22, 23, 24, 27, 28, 30, 39, 42, 44, 46, 49.
- the set comprising at least one platelet-miRNA does not comprise miRNA(s) selected from the group consisting of hsa-miR-126, hsa-miR-197, hsa-miR-223, hsa-miR-21 and hsa-miR-24.
- the invention in a third aspect, relates to a method for diagnosing a platelet-related disease in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to 56.
- the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a Paxgene-like tube, particularly preferred it is collected in a PAXgeneTM Blood RNA tube.
- the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the expression profile is determined from a whole blood sample in front of a background of white blood cells and red blood cells.
- the expression profile is determined from a whole blood sample without prior isolation (or extraction) of the platelet fraction
- the method of the third aspect of the present invention including the aforementioned embodiments comprises the following steps :
- the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2, more preferably said at least one miRNA is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 32, 34, 37, 38, 40, 42, 43, 44, 46, 47, 49, 51, 52.
- the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 14, 15, 17, 18, 21, 22, 24, 25, 27, 32, 35.
- the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 6, 7, 8, 10, 11, 15, 17, 18, 19, 23.
- the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 11 , 13, 16, 20, 21 , 22, 25, 26, 27, 28, 30, 31, 36, 41, 45.
- the disease is Multiple Sclerosis and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 6, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 5, 11 , 12, 13, 16, 19, 20, 21, 23, 24, 25, 26, 28, 29, 31, 32, 37, 39, 50, 55.
- the disease is Benign Prostate Hyperblasia (BPH) and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 7, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 16, 17, 18, 19, 21, 37, 40, 41.
- BPH Benign Prostate Hyperblasia
- the disease is Pancreatitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 8, more preferably said at least one miRNA is selected from the group SEQ ID NO: 12, 23, 26, 27, 29, 33, 35.
- the disease is Wilms' Tumor and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 9, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 30, 41, 46, 53.
- the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 6, 7, 8, 9, 11 , 12, 14, 19, 22, 26.
- the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 11, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 22, 23, 35, 39, 56.
- the disease is Pancreatic Cancer and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 12, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 12, 27, 29, 33.
- the disease is Periodontitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 13, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 9, 10, 1 1 , 12, 14, 15, 16, 20, 21, 23, 24, 25, 29, 30, 33, 34, 39.
- the disease is COPD and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 14, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 10, 13, 14, 16, 17, 18, 26, 36, 42, 43, 48.
- the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 5, 8, 9, 13, 14, 19, 22, 28, 33, 34, 38, 54.
- the disease is Ovarian Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 16, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 5, 6, 1 1 , 13, 16, 17, 19, 21 , 22,
- the set comprising at least one platelet-miRNA does not comprise miRNA(s) selected from the group consisting of hsa-miR-126, hsa-miR-197, hsa-miR-223, hsa-miR-21 and hsa-miR-24.
- the invention relates to a method for monitoring the progression of a platelet- related disease in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to 56.
- the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a Paxgene-like tube, particularly preferred it is collected in a PAXgeneTM Blood RNA tube.
- the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the expression profile is determined from a whole blood sample in front of a background of white blood cells and red blood cells.
- the expression profile is determined from a whole blood sample without prior isolation (or extraction) of the platelet fraction
- the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2, more preferably said at least one miRNA is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 32, 34, 37, 38, 40, 42, 43, 44, 46, 47, 49, 51, 52.
- the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 14, 15, 17, 18, 21, 22, 24, 25, 27, 32, 35.
- the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 6, 7, 8, 10, 11, 15, 17, 18, 19, 23.
- the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 11 , 13, 16, 20, 21 , 22, 25, 26, 27, 28, 30, 31, 36, 41, 45.
- the disease is Multiple Sclerosis and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 6, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 5, 11 , 12, 13, 16, 19, 20, 21, 23, 24, 25, 26, 28, 29, 31, 32, 37, 39, 50, 55.
- the disease is Benign Prostate Hyperblasia (BPH) and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 7, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 16, 17, 18, 19, 21, 37, 40, 41.
- BPH Benign Prostate Hyperblasia
- the disease is Pancreatitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 8, more preferably said at least one miRNA is selected from the group SEQ ID NO: 12, 23, 26, 27, 29, 33, 35.
- the disease is Wilms' Tumor and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 9, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 30, 41, 46, 53.
- the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 6, 7, 8, 9, 11 , 12, 14, 19, 22, 26.
- the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 11 , more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 22, 23, 35, 39, 56.
- the disease is Pancreatic Cancer and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 12, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 12, 27, 29, 33.
- the disease is Periodontitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 13, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 9, 10, 1 1 , 12, 14, 15, 16, 20, 21, 23, 24, 25, 29, 30, 33, 34, 39.
- the disease is COPD and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 14, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 10, 13, 14, 16, 17, 18, 26, 36, 42, 43, 48.
- the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 5, 8, 9, 13, 14, 19, 22, 28, 33, 34, 38, 54.
- the disease is Ovarian Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 16, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 5, 6, 11 , 13, 16, 17, 19, 21 , 22, 23, 24, 27, 28, 30, 39, 42, 44, 46, 49.
- the set comprising at least one platelet-miRNA does not comprise miRNA(s) selected from the group consisting of hsa-miR-126, hsa-miR-197, hsa-miR-223, hsa-miR-21 and hsa-miR-24.
- the invention relates to a method for determining the platelet activity in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to 56.
- the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a Paxgene-like tube, particularly preferred it is collected in a PAXgeneTM Blood RNA tube. It is preferred that the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells. In a further alternative embodiment, the expression profile is determined from a whole blood sample in front of a background of white blood cells and red blood cells. In a still further alternative embodiment, the expression profile is determined from a whole blood sample without prior isolation (or extraction) of the platelet fraction
- the method of the fifth aspect of the present invention comprises the following steps :
- a non-optimal platelet activity may be a platelet activity that is above or below a certain threshold. Hence the platelet activity may be increased or decreased when compared to said reference. Therefore upon administering said subject to therapy, drugs or other therapeutic measures may be applied with the goal to optimize the platelet activity or to bring the platelet-activity to a normal level, which is not above or below a certain threshold.
- the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2, more preferably said at least one miRNA is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 32, 34, 37, 38, 40, 42, 43, 44, 46, 47, 49, 51, 52.
- the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 14, 15, 17, 18, 21, 22, 24, 25, 27, 32, 35.
- the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 6, 7, 8, 10, 11, 15, 17, 18, 19, 23.
- the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 7, 8, 9, 1 1, 13, 16, 20, 21, 22, 25, 26, 27, 28, 30, 31, 36, 41, 45.
- the disease is Multiple Sclerosis and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 6, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 5, 11 , 12, 13, 16, 19, 20, 21, 23, 24, 25, 26, 28, 29, 31, 32, 37, 39, 50, 55.
- the disease is Benign Prostate Hyperblasia (BPH) and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 7, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 16, 17, 18, 19, 21, 37, 40, 41.
- BPH Benign Prostate Hyperblasia
- the disease is Pancreatitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 8, more preferably said at least one miRNA is selected from the group SEQ ID NO: 12, 23, 26, 27, 29, 33, 35.
- the disease is Wilms' Tumor and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 9, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 30, 41, 46, 53.
- the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 6, 7, 8, 9, 11 , 12, 14, 19, 22, 26.
- the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 1 1 , more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 22, 23, 35, 39, 56.
- the disease is Pancreatic Cancer and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 12, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 12, 27, 29, 33.
- the disease is Periodontitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 13, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 9, 10, 1 1 , 12, 14, 15, 16, 20, 21, 23, 24, 25, 29, 30, 33, 34, 39.
- the disease is COPD and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 14, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 10, 13, 14, 16, 17, 18, 26, 36, 42, 43, 48.
- the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 5, 8, 9, 13, 14, 19, 22, 28, 33, 34, 38, 54.
- the disease is Ovarian Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 16, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 5, 6, 11 , 13, 16, 17, 19, 21 , 22, 23, 24, 27, 28, 30, 39, 42, 44, 46, 49.
- the set comprising at least one platelet-miRNA does not comprise miRNA(s) selected from the group consisting of hsa-miR-126, hsa-miR-197, hsa-miR-223, hsa-miR-21 and hsa-miR-24.
- the invention relates to a method for monitoring the efficacy of an anti-platelet therapy in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to 56.
- the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a Paxgene-like tube, particularly preferred it is collected in a PAXgeneTM Blood RNA tube.
- the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the expression profile is determined from a whole blood sample in front of a background of white blood cells and red blood cells.
- the expression profile is determined from a whole blood sample without prior isolation (or extraction) of the platelet fraction
- RNA isolation of the total RNA from said whole blood sample b. Isolation of the total RNA from said whole blood sample c. Determining an expression profile of a set comprising at least one platelet-miRNA from the total RNA isolated.
- a non-optimal efficacy of anti-platelet therapy may be an anti-platelet therapy that is above or below a certain threshold.
- the efficacy of the anti-platelet therapy may be increased or decreased when compared to said reference. Therefore upon administering said subject to therapy, drugs or other therapeutic measures may be applied with the goal to optimize the efficacy of the anti-platelet therapy or to bring the efficacy of the anti-platelet therapy to a normal level, which is not above or below a certain threshold.
- the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2, more preferably said at least one miRNA is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 32, 34, 37, 38, 40, 42, 43, 44, 46, 47, 49, 51, 52.
- the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 14, 15, 17, 18, 21, 22, 24, 25, 27, 32, 35.
- the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 6, 7, 8, 10, 11, 15, 17, 18, 19, 23.
- the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 1 1, 13, 16, 20, 21, 22, 25, 26, 27, 28, 30, 31, 36, 41, 45.
- the disease is Multiple Sclerosis and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 6, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 5, 11 , 12, 13, 16, 19, 20, 21, 23, 24, 25, 26, 28, 29, 31, 32, 37, 39, 50, 55.
- the disease is Benign Prostate Hyperblasia (BPH) and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 7, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 16, 17, 18, 19, 21, 37, 40, 41.
- BPH Benign Prostate Hyperblasia
- the disease is Pancreatitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 8, more preferably said at least one miRNA is selected from the group SEQ ID NO: 12, 23, 26, 27, 29, 33, 35.
- the disease is Wilms' Tumor and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 9, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 30, 41, 46, 53.
- the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 6, 7, 8, 9, 11 , 12, 14, 19, 22, 26.
- the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 1 1 , more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 22, 23, 35, 39, 56.
- the disease is Pancreatic Cancer and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 12, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 12, 27, 29, 33.
- the disease is Periodontitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 13, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 9, 10, 1 1 , 12, 14, 15, 16, 20, 21, 23, 24, 25, 29, 30, 33, 34, 39.
- the disease is COPD and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 14, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 10, 13, 14, 16, 17, 18, 26, 36, 42, 43, 48.
- the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 5, 8, 9, 13, 14, 19, 22, 28, 33, 34, 38, 54.
- the disease is Ovarian Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 16, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 5, 6, 11 , 13, 16, 17, 19, 21 , 22, 23, 24, 27, 28, 30, 39, 42, 44, 46, 49.
- the set comprising at least one platelet-miRNA does not comprise miRNA(s) selected from the group consisting of hsa-miR-126, hsa-miR-197, hsa-miR-223, hsa-miR-21 and hsa-miR-24.
- the invention relates to a method for determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to 56.
- the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a Paxgene-like tube, particularly preferred it is collected in a PAXgeneTM Blood RNA tube.
- the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the expression profile is determined from a whole blood sample in front of a background of white blood cells and red blood cells.
- the expression profile is determined from a whole blood sample without prior isolation (or extraction) of the platelet fraction
- the method of the seventh aspect of the present invention comprises the following steps :
- the determining of an expression profile of a set comprising at least one platelet-miRNA (selected from SEQ ID NO: 1 to 56) from the total RNA isolated according to the first aspect (diagnosing a platelet-related component of a disease), to the second aspect (diagnosing a disease), to the third aspect (diagnosing a platelet-related disease), to the fourth aspect (monitoring the progression of a disease), to the fifth aspect (determining the platelet activity of a disease), to the sixth aspect (monitoring the efficacy of an anti-platelet-therapy) or to the seventh aspect (determining an expression profile of a set comprising at least on platelet- miRNA) of the invention comprises the steps :
- step (b) optionally amplifying the cDNA of step (a)
- miRNA-specific or universal reverse transcription DNA-primers are used for reverse transcription in step (a).
- miRNA-specific reverse transcription primers are listed in column B of Figure 17, examples of universal reverse transcription primers are 01igo-d(T)-Primers or RT primers listed in column C of Figure 20.
- miRNA-specific forward primer and universal reverse primer or alternatively universal forward and universal reverse primer are used for optionally amplifying the cDNA in step (b).
- miRNA-specific forward primer and universal reverse primer are listed in column C and D of Figure 17, examples of universal forward and universal reverse primer are listed in column D and E of Figure 20.
- miRNA-specific forward primer and universal reverse primer or alternatively miRNA-specific forward and partially universal reverse primer are used for quantifying the optionally amplified cDNA in step (c).
- the quantifying in step (c) is performed by real-time PCR, nucleic acid hybridization (e.g. microarray) or sequencing (e.g. next generation sequencing) techniques.
- the quantifying in step (c) by real-time PCR is utilizing dual-labeled hydrolysis probes that make use of the 5 ' -3 ' exonuclease activity of polymerase (e.g. Taqman-probes) or DNA-intercalating dyes (e.g. SYBRgreen).
- Examples of miRNA-specific forward primer and universal reverse primer are listed in column C and D of Figure 17, examples of miRNA-specific forward and partially universal reverse primer are listed in column B and C of Figure 18; examples of dual-labeled hydrolysis probes are listed in column E of Figure 17.
- the determining of an expression profile of a set comprising at least one platelet-miRNA (selected from SEQ ID NO: 1 to 56) from the total RNA isolated according to the first aspect (diagnosing a platelet-related component of a disease), to the second aspect (diagnosing a disease), to the third aspect (diagnosing a platelet-related disease), to the fourth aspect (monitoring the progression of a disease), to the fifth aspect (determining the platelet activity of a disease), to the sixth aspect (monitoring the efficacy of an anti-platelet-therapy) or to the seventh aspect (determining an expression profile of a set comprising at least on platelet- miRNA) of the invention comprises the steps : (a) adding a DN A- fragment to the 3 '-end of the at least one platelet-miRNA comprised in the total RNA isolated (from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells), thereby forming non-naturally occurring RNA
- DNA- fragments of 1 to 150 nucleotides in length are added in step (a) to the 3 '-end of the miRNAs by ligation or by polymerase-based elongation.
- Examples of said DNA-fragments are listed in Figure 19 or column A of Figure 20.
- step (miRNA-specific or) universal reverse transcription DNA-primers are used for reverse transcription in step (b). Further, it is preferred that miRNA-specific forward primer and universal reverse primer or alternatively miRNA-specific forward and partially universal reverse primer are used for quantifying the optionally amplified cDNA in step (c). Examples of miRNA-specific reverse transcription primers are listed in column B of Figure 17, examples of universal reverse transcription primers are oligo-d(T)-Primers or RT primers listed in column C of Figure 20. It is preferred that the quantifying in step (c) is performed by real-time PCR, nucleic acid hybridization or sequencing (e.g. next generation sequencing) techniques.
- the quantifying in step (c) by real-time PCR is utilizing dual-labeled hydrolysis probes that make use of the 5 ' -3 ' exonuclease activity of polymerase (e.g. Taqman-probes) or DNA-intercalating dyes (e.g. SYBRgreen).
- polymerase e.g. Taqman-probes
- DNA-intercalating dyes e.g. SYBRgreen
- the determining of an expression profile of a set comprising at least one platelet-miRNA (selected from SEQ ID NO: 1 to 56) from the total RNA isolated according to the first aspect (diagnosing a platelet-related component of a disease), to the second aspect (diagnosing a disease), to the third aspect (diagnosing a platelet-related disease), to the fourth aspect (monitoring the progression of a disease), to the fifth aspect (determining the platelet activity of a disease), to the sixth aspect (monitoring the efficacy of an anti-platelet-therapy) or to the seventh aspect (determining an expression profile of a set comprising at least on platelet- miRNA) of the invention comprises the steps :
- RNA-RNA hybrids (a) adding a RNA- fragment to the 3 '-end of the at least one platelet-miRNA comprised in the total RNA isolated (from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells), thereby forming non-naturally occurring RNA-RNA hybrids
- RNA- fragments of 1 to 150 nucleotides in length are added in step (a) to the 3 '-end of the miRNAs preferably by poly(A)-tailing reaction.
- the reverse- transcription of step (b) is preferably with universal RT -primers, e.g. oligo-d(T)-primers.
- the quantifying in step (c) is preferably utilizing miRNA-specific forward and partially universal reverse primer, e.g. miRNA-specific forward and partially universal reverse primer as listed in column B and C of Figure 18.
- the invention relates the use of a whole blood collection tube in the method according to any of the first, second, third, fourth, fifth, sixth of seventh aspect of the present invention including all its embodiments.
- a whole blood collection tube in the use according to the eight aspect of the invention contains an additive for RNA stabilization, preferably optionally contians an additive for stabilization of intracellular RNA, more preferably in said use the whole blood collection tube is a PAXgeneTM Blood RNA tube.
- said additive for RNA-stabilization is selected from the group consisting of catrimox-14, tetradecyltrimethylammonium oxalate, RNA- later, RNAsin or RNAretain.
- a PAXgeneTM Blood RNA tube is employed in the use according to the eight aspect of the invention for determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the invention relates to a kit for use in the method according to any of the first, second, third, fourth, fifth, sixth of seventh aspect of the present invention including all of its embodiments.
- Said kit for use in the method according to any of the first, second, third, fourth, fifth, sixth of seventh aspect of the present invention comprises :
- the expression profile and the reference expression profile are obtained from said set of at least one platelet-miR As selected from the group consisting SEQ ID NO: 1 to 56 and wherein the expression profile and the reference expression profile are determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the reference may be contained in the data carrier of the kit.
- the reference may be a reference sample and/or a reference standard that is included in the kit and which is employed when performing the kit, e.g. in the determining of the expression profile.
- the kit comprises a data carrier.
- the data carrier is an electronic or a non-electronic data carrier, more preferably it is an electronic data carrier, such as a storage medium.
- the data carrier comprised in the kit comprises a guide for use of the kit in the method according to any of the first, second, third, fourth, fifth, sixth of seventh aspect of the present invention.
- This guide may include instructions for the doctor and/or the diagnostic laboratory that are involved in the method according to any of the first, second, third, fourth, fifth, sixth of seventh aspect of the present invention.
- the guide may include a reference according to the present invention.
- the data carrier further comprises tools for analysis and evaluation of the determined expression profile(s).
- tools may be any tools to assist the doctor and/or the diagnostic laboratory in the method according to any of the first, second, third, fourth, fifth, sixth of seventh aspect of the present invention.
- these tools are software-tools that assist in analysis of the determined expression profile(s) and/or assist in the subsequently diagnosis.
- the tools for analysis and evaluation may include a reference according to the present invention.
- the kit optionally comprises whole bood collection tubes, which are preferably selected from group consisting of EDTA- , Na-citrate-, ACD-, Heparin-, PAXgeneTM Blood RNA-, Tempus Blood RNA-tubes and optionally contains an additive for stabilizing the RNA-fraction.
- whole bood collection tubes which are preferably selected from group consisting of EDTA- , Na-citrate-, ACD-, Heparin-, PAXgeneTM Blood RNA-, Tempus Blood RNA-tubes and optionally contains an additive for stabilizing the RNA-fraction.
- the present invention is composed of the following items :
- Method for diagnosing a platelet-related or platelet-activated component of a disease by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- Method for diagnosing a disease by determining an expression profile of a set comprising at least one platelet-miR A from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- Method for diagnosing a platelet-related disease in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- Method for monitoring the progression of a platelet-related disease in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- Method for determining the platelet activity in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- Method for monitoring the efficacy of an anti-platelet therapy in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- Identifying if said subject is affected by one of the disease conditions (detailed in items 1 to 6 f Optionally administering said affected subject to therapy of said disease condition
- the disease condition is selected from the group consisting of platelet-related or platelet-activated component of a disease, disease, platelet- related disease, progression of a platelet-related disease, platelet activity or efficacy of an anti-platelet therapy.
- Method for determining an expression profile of a set comprising at least one platelet- miR A from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the platelet-miRNAs are selected from the group consisting of SEQ ID NO: 1 to 56.
- the method according to any of the items 1 to 10 wherein the whole blood sample is collected in a whole blood collection tube, optionally containing an additive for RNA stabilization, preferably optionally containing an additive for stabilization of intracellular RNA, more preferably the whole blood is collected in a PAXgeneTM Blood RNA tube.
- RNA-stabilization is selected from the group consisting of catrimox-14, tetradecyltrimethylammonium oxalate, RNA- later, RNAsin or RNAretain.
- a whole blood collection tube in the method according to any of the items 1 to 12 wherein the expression profile is determined from the total RNA isolated from the whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- the method or use according to any of the items 1 to 14, wherein the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2.
- the method or use according to any of the items 1 to 14 , wherein the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3.
- the method or use according to any of the items 1 to 14, wherein the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4.
- the method or use according to any of the items 1 to 14, wherein the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5.
- the method or use according to any of the items 1 to 14, wherein the disease is Multiple Sclerosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 6.
- the method or use according to any of the items 1 to 14, wherein the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10.
- the method or use according to any of the items 1 to 14, wherein the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miR As listed in Figure 11.
- the method or use according to any of the items 1 to 14, wherein the disease is Pancreatic Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 12.
- the method or use according to any of the items 1 to 14, wherein the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15.
- kits for use in the method according to any of the items 1 to 12 or 15 to 29, comprising : a) means for determining an expression profile of a set comprising at least one platelet-miRNA
- the expression profile and the reference expression profile are obtained from said at least one platelet-miRNAs selected from the group consisting SEQ ID NO: 1 to 56 and wherein the expression profile and the reference expression profile are determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- a PAXgeneTM Blood RNA tube for determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from the total RNA isolated from the whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
- platelet-miRNAs are selected from the group consisting of SEQ ID NO: 1 to 56.
- step (b) optionally amplifying the cDNA of step (a)
- step (a) The method according to item 34, wherein miRNA-specific or universal reverse transcription DNA-primers are used for reverse transcription in step (a).
- step (c) The method according to any of the items 34 to 35, wherein miRNA-specific forward primer and universal reverse primer or miRNA-specific forward and partially universal reverse primer are used for quantifying the optionally amplified cDNA in step (c).
- step (b) The method according to any of the items 34 to 37, wherein miRNA-specific forward primer and universal reverse primer or miRNA-specific forward and partially universal reverse primer are used for optionally amplifying the cDNA in step (b). 38 The method according to any of the items 34 to 38 wherein that the quantifying in step is performed by real-time PCR, nucleic acid hybridization or sequencing techniques.
- step (c) The method according to any of the items 39 to 41, wherein miRNA-specific forward primer and universal reverse primer or miRNA-specific forward and partially universal reverse primer are used for quantifying the optionally amplified cDNA in step (c).
- step (c) is performed by real-time PCR, nucleic acid hybridization or sequencing techniques.
- FIG. 2 SARCOIDOSIS : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet- related disease, for determining the platelet-activity and/or for monitoring the efficacy of antiplatelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Sarcoidosis.
- RENAL CANCER Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Renal Cancer.
- Figure 4 COLON CANCER : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Colon Cancer.
- PSORIASIS Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet- related disease, for determining the platelet-activity and/or for monitoring the efficacy of antiplatelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Psoriasis.
- MULTIPLE SCLEROSIS Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Multiple Sclerosis.
- BENIGN PROSTATIC HYPERBLASIA (BPH) Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Benign Prostatic Hyperblasia (BPH).
- BPH Benign Prostatic Hyperblasia
- PANCREATITIS Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Pancreatitis.
- WILMS' TUMOR Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Wilms' Tumor.
- PROSTATE CANCER Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Prostate Cancer.
- Figure 1 1 LUNG CANCER : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Lung Cancer.
- PANCREATIC CANCER Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Pancreatic Cancer.
- SEQ ID NO: sequence identification number
- miRNA identifier of the miRNA according to miRBase (www.mirbase.org)
- Healthy Control median gl median intensity
- PERIODONTITIS Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Periodontitis.
- COPD Chronic obstructive pulmonary disease
- Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is COPD.
- MELANOMA Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet- related disease, for determining the platelet-activity and/or for monitoring the efficacy of antiplatelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Melanoma.
- OVARIAN CANCER Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Ovarian Cancer.
- Figure 17 miRNA-specific DNA-primers (column B) used for reverse transcription (RT) of at least one platelet-miRNA with SEQ ID NO: 1 to 56 to non-naturally occurring cDNA; miRNA-specific forward and universal reverse primers (column C, D) for quantification and optionally amplification of at least one platelet-miRNA with SEQ ID NO: 1 to 56 employed for determining of an expression profile of a set comprising at least one platelet-miRNA; dual- labeled hydrolysis probes (Taqman-probes, column E) utilized for quantifying of at least one platelet-miRNA with SEQ ID NO: 1 to 56 by real-time PCR .
- column B miRNA-specific DNA-primers
- RT reverse transcription
- C, D miRNA-specific forward and universal reverse primers
- Taqman-probes, column E dual- labeled hydrolysis probes
- Figure 18 miRNA-specific forward primer (column B) and partially universal reverse primer (column C) for quantification and optionally amplification of cDNA-transcripts of at least one platelet-miRNA with SEQ ID NO: 1 to 56 employed for determining of an expression profile of a set comprising at least one platelet-miRNA.
- Figure 19 DNA- fragments added to the 3 '-end of at least one platelet-miRNA with SEQ ID NO: 1 to 56 employed for determining of an expression profile of a set comprising at least one platelet-miRNA, thereby forming non-naturally occurring RNA-DNA hybrids.
- EXAMPLE 1 Preparation of total RNA for determination of platelet-miRNAs from whole blood samples without prior depletion or removal of the white blood cells and/or the red blood cells
- RNA Blood of patients and healthy controls was drawn in PAXgeneTM Blood RNA tubes (PreAnalytiX GmbH, Hombrechtikon, Switzerland). For each blood donor, 2.5 ml of peripheral whole blood was collected. Herein, the blood cell pellet (the intra-cellular blood fraction comprising red blood cells, white blood cells and platelets) was collected at the bottom of the tube by centrifugation. For further processing the blood cell pellet was used, while the supernatant (including the extra-cellular blood fraction) was discarded. Total RNA, including the small RNA (miRNA- fraction) was isolated from the pelleted blood cells using the miRNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and the resulting RNA was stored at -70° C before use in expression profiling experiments.
- RNA-samples were analyzed employing microarray hybridization on the Geniom Realtime Analyzer (febit biomed GmbH, Heidelberg, Germany) using the Geniom Biochip miRNA homo sapiens.
- Geniom Realtime Analyzer fibit biomed GmbH, Heidelberg, Germany
- Each microfluidic microarray contains complementary dna-probes of 866 miRNAs and miRNA* (each represented by 7 replicates) as annotated in the Sanger miRBase 12.0.
- Sample labeling with biotin has been carried out by enzymatic on-chip labeling of miRNAs employing the MPEA-assay (Vorwerk et.al. N Biotechnol. 2008, 25(2-3): 142-9).
- the biochip was washed automatically and a program for signal enhancement was processed with the Geniom Realtime Analyzer. The resulting detection pictures were evaluated using the Geniom Wizard Software. For each array, the median signal intensity was extracted from the raw data file such that for each miRNA seven intensity values have been calculated corresponding to each replicate copy of miRBase on the array. Following background correction, the seven replicate intensity values of each miRNA were summarized by their median value. To normalize the data across different arrays, quantile normalization was applied and all further analyses were carried out using the normalized and background subtracted intensity values.
- the remaining miRNA-biomarker candidates were aligned to a list of selected platelet- miRNAs described to be highly expressed in platelets (PMID 21415270, 22371016, 23323973), wherein the list of highly expressed miRNAs contained the 30% highest expressed platelet- miRNAs.
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Abstract
The present invention relates to methods, kits and uses for determining platelet-miRNAsin a whole blood sample from a subject.
Description
DETERMINATION OF PLATELET-MIRNAS
TECHNICAL FIELD OF THE INVENTION
The present invention relates to methods, use and kits for the determination of platelet-miRNAs
BACKGROUND OF THE INVENTION
Today, biomarkers play a key role in early diagnosis, risk stratification, and therapeutic management of various diseases. MicroRNAs (miRNAs) are a new class of biomarkers. They represent a group of small noncoding RNAs that regulate gene expression at the posttranslational level by degrading or blocking translation of messenger RNA (mRNA) targets. So far, miRNAs have been extensively studied in tissue material where it was found that miRNAs are expressed in a highly tissue-specific manner. Since recently it is known that miRNAs are not only present in tissues but also in body fluid samples, including blood. Nevertheless, the mechanism why miRNAs are found in blood, especially in the cellular blood fraction (blood cells of subfractions thereof) or in the extra-cellular fraction (serum/plasma), or their function in these blood fractions is not understood yet.
The inventors of the present invention assessed for the first time the expression of platelet- miRNAs from whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells, hence in front of a background of white blood cells and/or red blood cells. They surprisingly found that especially the platelet-miRNAs are significantly dysregulated in whole blood samples collected in whole blood collection tubes, preferably collected in Paxgene-like tubes, more preferably collected in PAXgene™ Blood RNA tubes. This is surprising since the RNA-content of the anucleated platelets is approximately 10.000 times lower when compared to the RNA-content of the nucleated white blood cells, with which the platelets are mixed in the whole blood sample collected in said whole blood collection tubes. Surprisingly, the expression of the platelet-miRNAs can be determined directly from total RNA isolated from the mixture of platelets, white blood cells and red blood cells present in the whole blood collected in said whole blood collection tubes without the need for prior depletion or removal of the white blood cells and/or the red blood cells. Hence the expression of the platelet- miRNAs can be determined in front of a background of white blood cells and/or red blood cells, without the need to isolate (or enrich) the platelets or the platelet fraction from the whole blood sample before. This is of advantage to applications, especially diagnostic applications, where the determination of said platelet-miRNAs can be directly determined from a whole blood sample without the need for additional sample processing steps (e.g. removal or depletion of the red
blood cells and/or white blood cell fractions, or isolation of the platelet fraction). This results in easier, quicker, cheaper preanalytic processing of such samples, which is especially valuable for competitive diagnostic applications. The inventors of the present invention further surprisingly found that platelet-miR As that are determined from total R A isolated from whole blood samples without prior depletion and/or removal of white blood cells and/or red blood cells (or in front of a background of white blood cells and/or red blood cells; or without prior isolation of the platelet fraction), may be employed as biomarkers for non-invasive diagnosis of diseases, platelet-related diseases or platelet-related (platelet-activated) inflammatory components of such diseases. Furthermore, it was found that platelet-miRNAs that are determined from total RNA isolated from whole blood samples without prior depletion and/or removal of white blood cells and/or red blood cells (or in front of a background of white blood cells and/or red blood cells; or without prior isolation of the platelet fraction) may be employed as biomarkers for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy. Therefore, the inventors of the present invention found that the whole blood collection tubes, preferably the Paxgene-like tubes, more preferably the PAXgene™ Blood RNA tubes, are suitable or useful for determination of expression profiles of platelet-miRNAs and that such expression profiles may be used in a method for non-invasive diagnosis of diseases, for non-invasive diagnosis of platelet-related diseases or for non-invasive diagnosis of platelet-related (platelet-activated) inflammatory components of such diseases and for determining platelet-activity or for monitoring the efficacy of anti-platelet therapy.
SUMMARY OF THE INVENTION
In a first aspect, the invention provides for a method for diagnosing a platelet-related or a platelet-activated component of a disease by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
In a second aspect, the invention provides for a method for diagnosing a disease by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
In a third aspect, the invention provides for a method for diagnosing a platelet-related disease in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a
whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
In a fourth aspect, the invention provides for a method for monitoring the progression of a platelet-related disease in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
In a fifth aspect, the invention provides for a method for determining the platelet activity in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
In a sixth aspect, the invention provides for a method for monitoring the efficacy of an antiplatelet therapy in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
In a seventh aspect, the invention provides for a method for determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
In an eighth aspect, the invention provides for the use of a whole blood collection tube in the method according to any of the first, second, third, fourth, fifth, sixth or seventh aspect of the invention, wherein the expression profile of a set of at least one platelet-miRNA is determined from the total RNA isolated from the whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
In a ninth aspect, the invention provides for a kit for use in the method in the method according to any of the first, second, third, fourth, fifth, sixth or seventh aspect of the invention, comprising:
a) means for determining an expression profile of a set comprising at least one platelet-miRNA
b) a reference derived from at least one reference expression profile
c) optionally a data carrier
d) optionally a whole blood collection tube
wherein the expression profile and the at least one reference expression profile are obtained from said at least one platelet-miRNA selected from the group consisting SEQ ID NO : 1 to 56 and wherein the expression profile and the at least one reference expression profile are determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
This summary of the invention does not necessarily describe all features of the invention.
DETAILED DESCRIPTION OF THE INVENTION Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.
Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).
To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, and recombinant DNA techniques are employed which are explained in the literature in the field (cf, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in
their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
As used in this specification and in the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise. For example, the term "a test compound" also includes "test compounds".
The terms "micro RNA" or "miRNA" refer to single-stranded RNA molecules of at least 10 nucleotides and of not more than 35 nucleotides covalently linked together. The miRNAs regulate gene expression and are encoded by genes from whose DNA they are transcribed, but miRNAs are not translated into protein (i.e. miRNAs are non-coding RNAs). The terms "micro RNA*" or "miRNA*" refer to miRNA molecules derived from the passenger strand upon processing. In the context of the present invention, the terms "miRNA" and "miRNA*" are interchangeable used. The miRBase (www.mirbase.org) is a well established repository and searchable database of published miRNA sequences and annotation. Because of the conservation of miRNAs among species, for example between humans and other mammals, e.g. animals such as mice, monkey or rat, a human miRNA may also be suitable for detecting the respective miRNA orthologue(s) in another species, e.g. in another mammal, e.g. in an animal such as mouse or rat or vice versa.
The term "platelet" as used in the context of the present invention refers to the smallest type of blood cells, also known as "thrombocytes", which are released into the blood stream from bone marrow megakaryocytes. Platelets play a crucial role in haemostasis and thrombosis. Platelets do not contain a nucleus, therefore missing substantial parts of the microRNA machinery components required for transcription and nuclear processing. Platelets comprise approximately 10.000 times less RNA when compared to nucleated cells (e.g. white blood cells). Platelets contain miRNAs (PMID 21415270, 22371016, 23323973), but it is not yet completely understood where these originate from and how these interact in translational control or repression of platelet mRNAs.
The term "platelet-miRNA" as used in the context of the present invention refers to a miRNA that is expressed in platelets (thrombocytes). This does not necessarily mean that this platelet- miRNA is exclusively expressed in platelets and not in any other blood cells, in other cells, in other body fluids or in tissue. Currently, there are approximately 400 miRNAs described to be expressed in platelets. The platelet-miRNAs according to the present invention are selected from
the group consisting of SEQ ID NO: 1 to SEQ ID NO: 56 and are listed in Figure 1. According to the present invention the expression level of said platelet-miRNAs selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO : 56 are in a range that allows for direct determination from a whole blood sample without the need for additional sample processing steps (e.g. removal or depletion of the red blood cells and/or white blood cell fractions, or isolation of the platelet fraction), which is of advantage to commercial applications, especially in the field of diagnostics. Hence, the platelet-miRNAs selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 56 can be determined directly from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells. This allows to make the biomarker information contained in said platelet-miRNAs directly available without prior removal or depletion of the red blood cells and/or white blood cells. Furthermore, besides the criteria of being reproducibly expressed in platelets and showing sufficient expression level, the inventors of the present invention applied further criteria based on expression data in a plurality of diseases for selection of said 56 platelet-miRNAs with SEQ ID NO: 1 to SEQ ID NO: 56 from the approximately 400 miRNAs that are described to be expressed in platelets. The inventors investigated a plurality of diseases, including Cancer (e.g. Renal Cancer, Colon Cancer, Lung Cancer, Melanoma, Ovarian Cancer, Wilm's Tumor, Prostate Cancer), Autoimmune Diseases (e.g. Sarcoidosis, Psoriasis, Multiple Sclerosis), Neurodegenerative Diseases (e.g. Multiple Sclerosis), Inflammatory Diseases (Pancreatitis, BPH, COPD, Periodontitis), including appropriate references and selected said 56 platelet-miRNAs with SEQ ID NO: 1 to SEQ ID NO: 56 from the resulting expression data (e.g .see Figure 2, 4, 5, 6, 7 ,8 ,9 ,10, 11, 12, 13 ,14, 15, 16,). For selection a 1.5 -fold threshold of differential expression between the disease and the reference was employed.
The term "whole blood sample", as used in the context of the present invention, refers to a blood sample originating from a subject containing all blood fractions, including both the cellular (red blood cells, white blood cells, platelets) and the extra-cellar blood fraction (serum, plasma). The "whole blood sample" may be derived by removing blood from a subject by conventional blood collecting techniques, but may also be provided by using previously isolated and/or stored blood samples. Preferably, the whole blood sample from a subject (e.g. human or animal) has a volume of between 0.1 and 40 ml, more preferably of between 0.5 and 20 ml, more preferably between 1 and 15 ml and most preferably between 2 and 10 ml, i.e. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 51, 12, 13, 14, 15, 16, 17, 18, 19, 20 ,21 , 22, 23, 24, 25, 26 ,27, 28, 29 ,30 ,31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 ml.
The term "whole blood collection tube" as used in the context of the present invention relates to blood collection tube, that is used for collection of whole blood samples, preferebly a whole
blood collection tube according to the present invention is a Paxgene-like tube, a PAXgene™ Blood RNA tube, a Tempus Blood RNA tube, an EDTA-tube, a Na-citrate tube or a ACD-tube (Acid citrate dextrose). Preferably, when the whole blood sample is collected, the RNA-fraction, -especially the miRNA fraction- may be protected/guarded against degradation. For this purpose special whole blood collection tubes (e.g. PAXgene™ Blood RNA tubes from Preanalytix, Tempus™ Blood RNA tubes from Applied Biosystems) or additives (e.g. RNAlater™ from Ambion, RNAsin® from Promega, RNA Retain® from Asuragen, catrimox-1 4 o r tetradecyltrimethyl-ammonium oxalate or derivatives thereof) that stabilize the RNA fraction and/or the miRNA fraction, may be employed.
The term "Paxgene-like tube" as used in the context of the present invention relates to blood collection tubes, which are suited or used for collection of whole blood samples, which contain additives for stabilization of RNA and which are suited for expression analyses of the intracellular RNA and/or intracellular miRNAs and/or platelet-miRNAs. Prefereably, herein additives for stabilization of RNA are employed, including, but are not limited to, RNAlater™, RNAsin®, RNA Retain® catrimox-14 or tetradecyltrimethylammonium oxalate or derivatives thereof. It is particularly preferred that "Paxgene-like tubes" are "PAXgene™ Blood RNA tubes", which are suited for collection of whole blood samples and which contain additives for cell lysis and stabilization of intracellular RNA and which are suited for expression analyses of RNAs and/or miRNAs and/or platelet-miRNAs. Preferably, herein additives for cell lysis and stabilization of intracellular RNA include, but are not limited to, catrimox-1 4 or tetradecyltrimethylammonium oxalate or derivatives thereof. PAXgene™ Blood RNA tubes are currently marketed by PreAnalytix ( w w w . p re a n a I yt i x . c o m ) . Further details on the PAXgene™ tubes (manuals, application notes etc.), its technology and how to use these can be found at the websites of Preanalytix (www.preanalytix.com/product-catalog/blood/), Qiagen (www.qiagen.com products/catalog /sample-technologies/rna-sampie-technoiogies/) or Becton Dickinson (www.bd.com/vacutainer/ products/molecular/) . Further details rega rd i ng the PAXgene™ technology, especially, regarding said additives for cell lysis and stabilization of intracellular RNA are detailed in US6602718, US6617170, US6821789, US6681213, US 7270953, US 7682790, WO02/00599, W094/18156, US 5985572, US5010183, which are all incorporated here by reference in their entirety. It is understood that any other blood collection tube, which is suited for collection of whole blood samples, which contain additives for stabilization of intracellular RNA and which are suited for expression analyses of intracellular RNA and/or intracellular miRNAs, and/or platelet-miRNAs, also fall under the term "Paxgene- like tube" as used in the context of the present invention.
The term "total RNA" as used herein relates to the RNA isolated from a whole blood sample comprising the miRNA- fraction. The total RNA, comprising the miRNA- fraction or comprising a miRNA-enriched fraction, is obtained by lysis (e.g. Trizol) of the blood cells of the whole blood sample, followed by RNA isolation (extraction) e.g. by phenol/chloroform extraction and/or separation based techniques (e.g. glass fibre filter column, silica-membrane column). Examples of kits for RNA isolation and purification include the miRNeasy Kits (Qiagen), PAXgene™ Blood miRNA Kit (Qiagen), mirVana PARIS Kit (Life Technologies), PARIS Kit (Life Technologies), Tempus Spin RNA Isolation Kit (Life Technologies). Preferably, the total RNA according to the present invention contains the miRNA- fraction or contains a miRNA- enriched fraction.
The term "intracellular RNA" as used herein relates to the RNA present in the blood cells (platelets, white blood cells, red blood cells) of a whole blood sample, comprising the miRNA- fractions.
The term "expression profile" as used in the context of the present invention, represents the a measure that correlates with the miRNA expression (level) in a sample. By determining the miRNA expression profile, each miRNA is represented by a numerical value. The higher the value of an individual miRNA, the higher is the expression level of said miRNA, or the lower the value of an individual miRNA, the lower is the expression level of said miRNA. The expression profile may be generated by any convenient means, e.g. nucleic acid hybridization (e.g. to a microarray), nucleic acid amplification (PCR, RT-PCR, qRT-PCR, high-throughput RT-PCR), ELISA for quantitation, next generation sequencing (e.g. ABI SOLID, Illumina Genome Analyzer, Roche/454 GS FLX), flow cytometry (e.g. LUMINEX, Milipore Guava) and the like, that allow the determination of a miRNA expression profile in a subject and comparison between samples. The sample material measured by the aforementioned means are derived from a whole blood sample (without prior depletion or removal of the white blood cells and/or the red blood cells or without isolation of the platelet fraction or in front of a background of white blood cells and/or red blood cells), comprising said platelet-miRNAs and may be total RNA, labeled total RNA, amplified total RNA, cDNA, labeled cDNA, amplified cDNA, miRNA, labeled miRNA, amplified miRNA or any derivatives that may be generated from the aforementioned RNA/DNA species. The "expression profile", as used herein, relates to a collection of expression (levels) of at least one miRNAs, preferably of least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 51 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 ,39 ,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or 56 or more miRNAs.
The term "determining an expression profile" as used herein, relates to the determination of the expression profile of set comprising at least one platelet-miRNA, hence from miRNA(s)
expressed in platelets, preferably from miRNAs expressed in platelets selected from the group consisting of SEQ ID NO 1 to 56. By doing so, the determination of the expression profile is a measure that directly or indirectly correlates with the levels of platelet-miRNAs present in said whole blood sample. Herein, all steps or transformations required to bring the isolated total RNA (comprising the platelet-miRNAs) into a form which allows to determine the expression profile by any convenient means (e.g. nucleic acid hybridisation, nucleic acid amplification, polymerase extension, mass spectroscopy, flow cytometry, sequencing) and which are known to the person skilled in the art, are included, e.g. miRN A- isolation, RNA- or miRNA-enrichment, RNA- or miRNA-purification, RNA- or miRNA-labeling, polymerase extension of RNA or miRNA, ligation of RNA or miRNA, reverse-transcription of RNA or miRNA into cDNA, amplification of the cDNA, labelling of cDNA.
The term "nucleic acid hybridization", as used herein, relates to a means for determining an expression profile. The nucleic acid hybridization may be performed using a microarray/biochip or in situ hybridization. For nucleic acid hybridization, for example, the polynucleotides (probes) with complementarity to the corresponding platelet-miRNAs to be detected are e.g. attached to a solid phase to generate a microarray/biochip. Said microarray/biochip is then incubated with a sample containing the platelet-miRNA(s) (or a species that is derived from said platelet- miRNA(s)), which may be labelled or unlabelled. Quantification of the expression level of the miRNAs may then be carried out e.g. by direct read out of said label or by additional manipulations, e.g. by use of an enzymatic reaction. Alternatively, the polynucleotides which are at least partially complementary to miRNAs having SEQ ID NO: 1 to 56 (or a species derived thereof, e.g. a cDNA-species) are contacted with said sample containing said platelet-miRNA(s) (or a species that is derived from said platelet-miRNA(s), e.g. a cDNA-species) in solution to hybridize. Afterwards, the hybridized duplexes are pulled down to the surface and successfully captured miRNAs are quantitatively determined (e.g. FlexmiR-assay, FlexmiR v2 detection assays from Luminex, Fireplex assay from Firefly Bioworks).
The term "nucleic acid amplification", as used herein, relates to a means for determining an expression profile. Nucleic acid amplification may be performed using real time polymerase chain reaction (RT-PCR) such as real time quantitative polymerase chain reaction (RT qPCR). The standard real time polymerase chain reaction (RT-PCR) is preferred for the analysis of a single miRNA or a set comprising a low number of miRNAs (e.g. a set of at least 2 to 10 miRNAs), whereas high-throughput RT-PCR technologies (e.g. OpenArray from Applied Biosystems, SmartPCR from Wafergen, Biomark System from Fluidigm) are also able to measure large sets (e.g a set of 5, 10, 20, 30, 50, 80, 100, 200 or more) of miRNAs in a high parallel fashion. The aforesaid real time polymerase chain reaction (RT-PCR) may include the
following steps: (i) extracting the total RNA from a whole blood sample collected in a whole blood collection tube, preferably collected in a Paxgene-like tube, particularly preferably collected in a PAXgene™ Blood RNA tube, obtained from a subject, (ii) obtaining cDNA samples by RNA reverse transcription (RT) reaction using universal or miRNA-specific primers; (iii) optionally pre-amplifying the cDNA of step (ii) via polymerase chain reaction (PCR), (iv) amplifying the optionally pre-amplified cDNA via polymerase chain reaction (PCR), thereby monitoring the amplification through a previously added fluorescent reporter dye (e.g. SYBR Green) or fluorescent reporter probe (e.g. Taqman probe), and (v) detecting the miRNA(s) level in the sample from the monitoring in step (iv). In Step (i) the isolation and/or extraction of RNA may be omitted in cases where the RT-PCR is conducted directly from the miRNA-containing sample. Kits for determining a miRNA expression profile by real time polymerase chain reaction (RT-PCR) are e.g. from Life Technologies, Applied Biosystems, Ambion, Roche, Qiagen, Invitrogen, SABiosciences, Exiqon.
The term "sequencing", as used herein, relates to a means for determining an expression profile, including conventional (Maxam-Gilbert, Sanger) sequencing technology, Pyrosequencing or next generation sequencing technology (e.g. ABI SOLID, Illumina Hiseq, Gnubio, Pacific Biosystems, Roche 454) or any other sequencing technology, capable of determination of the expression profile of set comprising at least one platelet-miRNA.
The term "reference" as used in the context of the present invention refers to a reference to which the expression profile of a test sample of a subject is compared in the course of noninvasive diagnosis of diseases, platelet-related diseases or platelet-related (platelet-activated) inflammatory components of such diseases or in the course of determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy. Herein, both the expression profile of the subject to be tested as well as the reference, are determined from the same platelet-miRNAs and the same sample type (collected and worked up in the same way), preferably they are determined from a whole blood sample collected in whole blood collection tubes, preferably collected in Paxgene-like tubes, more preferably collected in PAXgene™ Blood RNA tubes. The reference may be a reference expression profile obtained from determining one or more expression profiles of set comprising at least one platelet-miRNA from one or more whole blood samples collected in whole blood collection tubes, preferably determined from one or more whole blood samples collected in Paxgene-like tubes, more preferably determined from one or more whole blood samples collected in a PAXgene™ Blood RNA tubes, in one or more reference subjects. Furthermore, the reference may be an algorithm, a mathematical function or a score that was developed from such an aforementioned reference expression profile.
The term "diagnosing" as used in the context of the present invention refers to the process of determining a possible disease or disorder or a certain component of such a disease (e.g. an inflammatory component, a neuroinflammatory component of a disease) and therefore is a process attempting to define the (clinical) condition of a subject. The determination of the expression profile of at least one platelet-miR A according to the present invention correlates with the (clinical) condition of said subject. Preferably, the diagnosis comprises (i) determining the occurrence/presence of the disease ( or of a component of a disease), especially in an (very) early phase of the disease (ii) monitoring the course or progression of the disease, (iii) staging of the disease, (iv) measuring the response of a patient affected with the disease to therapeutic intervention, (v) monitoring the efficacy of a therapeutic intervention and/or (vi) segmentation of a subject suffering from the disease.
The term "platelet-related component of a disease" as used in the context of the present invention refers to an aspect or feature of the disease that is platelet-related or related to the function/dis function of platelets in said disease. Preferably, such platelet-related components include, but are not limited to, haemostasis-related, thrombosis-related, immunity-related, or inflammation-related aspects or features of the disease, more preferably the platelet-related component of a disease is an inflammatory component of the disease.
The term "platelet-activated component of a disease" as used in the context of the present invention refers to an aspect or feature of the disease that is platelet-activated or activated due to function/dis function of platelets in said disease. Preferably, such platelet-activated components include, but are not limited to, haemostasis-activated, thrombosis-activated, immunity-activated, or inflammation-activated aspects or features of the disease, more preferably the platelet- activated component of a disease is an inflammatory component of the disease.
An overview of the platelet-miR As well suited in the method, use or kit according to the present invention are the platelet-miRNAs with SEQ ID NO: 1 to 56, which are listed in Figure 1.
An exemplarily approach to determine expression profiles of a set comprising at least one platelet-miRNA selected from the group consisting of SEQ ID NO: 1 to 56 starting from whole blood collected in whole blood collection tubes, preferably collected in Paxgene-like tubes, particularly preferably collected in PAXgene™ Blood RNA tubes, without prior depletion or removal of white blood cells and/or red blood cells (or without prior isolation of the platelet fraction or in front of a background of white blood cells and/or red blood cells) -exemplarily starting from whole blood collected in PAXgene™ Blood RNA-tubes- is summarized below : Step 1 : Providing a whole blood sample of a subject : Whole blood is drawn from a subject into a PAXgene™ Blood RNA tube (or into another whole blood collection tube, preferably
into a Paxgene-like tube), drawing of 2.5 ml of whole blood into a PAXgene™ Blood RNA tube is sufficient for the downstream analyses. The tube should be carefully inverted to ensure that the reagents contained are thoroughly mixed with the blood. The tube maybe stored before analysis (e.g. at 4°C for up to 3 days, at -80°C for up to several months).
Step 2: Isolation of the total RNA from said whole blood sample : The PAXgene™ Blood RNA tube, which was allowed to incubate at room temperature for at least 1 hour, is centrifuged to form a blood cell pellet (comprising white blood cells, red blood cells, platelets) at the bottom of the tube, which is collected, whereas the supernatant is discarded. The total RNA (comprising the miRNA fraction) is isolated from the collected blood cell pellet (comprising white blood cells, red blood cells, platelets) using suitable kits (e.g. miRNeasy kit) and/or purification methods.
Step 3: Determining an expression profile of a set comprising at least one platelet-miRNA from the total RNA isolated : From the total RNA isolated the expression profile of at least one platelet-miRNA, selected from the group consisting of SEQ ID NO: 1 to SEQ ID
NO: 56 is measured using experimental techniques. These techniques include but are not limited to nucleic acid hybridisation based approaches, nucleic acid amplification methods (PCR, RT-PCR, qPCR), sequencing, next generation sequencing, flow cytometry and/or mass spectroscopy. In a preferred embodiment, it may be required in Step 3 that the total RNA or that individual platelet-miRNAs are reverse-transcribed into cDNA and optionally be amplified before the expression profile is determined. In order to make use of the determined expression profiles in a method for diagnosing a disease and/or for diagnosing of a platelet- related (or platelet-activated) inflammatory component of a disease and/or for diagnosing a platelet-related disease and/or for monitoring the progression of a platelet-related (platelet-activated) disease and/or for determining the platelet activity and/or for monitoring the efficacy of an anti-platelet therapy, further steps are performed :
Step 4: Comparing said expression profile to a reference : The expression profile obtained in Step 3) is compared to a reference. The reference may be e.g. a reference expression profile, obtained from determining one or more expression profile of said at least one platelet-miRNA from whole blood collected in the same type of whole blood collection tube - exemplarily here collected in a PAXgene™ Blood RNA tube- as in Step 1 (or collected in a another whole blood collection tube, preferably collected into a Paxgene- like tube as in Step 1) in one or more reference subjects or the reference may be an algorithm, a mathematical function or a score that was developed from such a reference expression profile.
Step 5 : Identifying if said subject is affected by one of the disease conditions : The comparison to the reference then allows to identify if said subject is affected or not affected by the disease (and/or the platelet- related (or platelet-activated) inflammatory component of the disease and/or the platelet-related disease) or alternatively if certain thresholds for monitoring the progression of a platelet-related (platelet-activated) disease and/or if thresholds for determining the platelet activity and/or if thresholds for monitoring the efficacy of an anti-platelet therapy are reached.
Step 6: Optionally administering said affected subject to therapy of said condition : Optionally, said subject, that was identified to be affected by the disease (and/or the platelet- related (or platelet-activated) inflammatory component of the disease and/or the platelet-related disease), is administered to therapy, e.g. by treating the subject with drugs suited for therapy of said disease (and/or drugs suited for said the platelet- related (or platelet- activated) or said inflammatory component of the disease and/or drugs suited for said platelet-related disease). Or alternatively subjecting said subject, if certain thresholds for monitoring the progression of a platelet-related (platelet-activated) disease and/or if thresholds for determining the platelet activity and/or if thresholds for monitoring the efficacy of an anti-platelet therapy are reached, to an altered therapeutic scheme, wherein said altered therapeutic scheme may be an increased or decreased therapeutic scheme with administering appropriate drugs at increased or decreased dosage.
In a first aspect, the present invention relates to method for diagnosing a platelet-related (or platelet-activated) component of a disease by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
It is preferred that in said method the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to 56.
It is preferred that the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a Paxgene-like tube, particularly preferred it is collected in a PAXgene™ Blood RNA tube.
It is preferred that that the platelet-related (or platelet-activated) component of a disease is a inflammatory component of said disease.
It is preferred that the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells. In a further alternative embodiment, the expression profile is determined from a whole blood sample in front of a
background of white blood cells and red blood cells. In a still further alternative embodiment, the expression profile is determined from a whole blood sample without prior isolation (or extraction) of the platelet fraction
Preferably, the method of the first aspect of the present invention including the aforementioned embodiments comprises the following steps :
a. Providing a whole blood sample of a subject
b. Isolation of the total R A from said whole blood sample
c. Determining an expression profile of a set comprising at least one platelet-miRNA from the total RNA isolated.
d. Comparing said expression profile to a reference
e. Identifying if said subject is affected by the disease
f. Optionally administering said affected subject to therapy suitable for said disease
In a still further embodiment, the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2, more preferably said at least one miRNA is selected from SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 32, 34, 37, 38, 40, 42, 43, 44, 46, 47, 49, 51, 52.
In a still further embodiment, the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 14, 15, 17, 18, 21, 22, 24, 25, 27, 32, 35.
In a still further embodiment, the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 6, 7, 8, 10, 11, 15, 17, 18, 19, 23.
In a still further embodiment, the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 7, 8, 9, 1 1, 13, 16, 20, 21, 22, 25, 26, 27, 28, 30, 31, 36, 41, 45.
In a still further embodiment, the disease is Multiple Sclerosis and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 6, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 5, 1 1, 12, 13, 16, 19, 20, 21, 23, 24, 25, 26, 28, 29, 31, 32, 37, 39, 50, 55.
In a still further embodiment, the disease is Benign Prostate Hyperblasia (BPH) and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 7, more
preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 16, 17, 18, 19, 21, 37, 40, 41.
In a still further embodiment, the disease is Pancreatitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 8, more preferably said at least one miRNA is selected from the group SEQ ID NO: 12, 23, 26, 27, 29, 33, 35.
In a still further embodiment, the disease is Wilms' Tumor and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 9, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 30, 41, 46, 53.
In a still further embodiment, the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 9, 1 1, 12, 14, 19, 22, 26.
In a still further embodiment, the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 11, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 22, 23, 35, 39, 56.
In a still further embodiment, the disease is Pancreatic Cancer and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 12, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 12, 27, 29, 33.
In a still further embodiment, the disease is Periodontitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 13, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 9, 10, 1 1, 12, 14, 15, 16, 20, 21, 23, 24, 25, 29, 30, 33, 34, 39.
In a still further embodiment, the disease is COPD and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 14, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 10, 13, 14, 16, 17, 18, 26, 36, 42, 43, 48.
In a still further embodiment, the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 5, 8, 9, 13, 14, 19, 22, 28, 33, 34, 38, 54.
In a still further embodiment, the disease is Ovarian Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 16, more preferably said at
least one miRNA is selected from the group SEQ ID NO: 1 , 2, 5, 6, 1 1 , 13, 16, 17, 19, 21 , 22, 23, 24, 27, 28, 30, 39, 42, 44, 46, 49.
In a still further embodiment of the first aspect of the present invention, the set comprising at least one platelet-miRNA does not comprise miRNA(s) selected from the group consisting of hsa-miR-126, hsa-miR-197, hsa-miR-223, hsa-miR-21 and hsa-miR-24.
In a second aspect, the invention relates to a method for diagnosing a disease by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
It is preferred that in said method the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to 56.
It is preferred that the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a Paxgene-like tube, particularly preferred it is collected in a PAXgene™ Blood RNA tube.
It is preferred that the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells. In a further alternative embodiment, the expression profile is determined from a whole blood sample in front of a background of white blood cells and red blood cells. In a still further alternative embodiment, the expression profile is determined from a whole blood sample without prior isolation (or extraction) of the platelet fraction
Preferably, the method of the second aspect of the present invention including the aforementioned embodiments comprises the following steps :
a. Providing a whole blood sample of a subject
b. Isolation of the total RNA from said whole blood sample
c. Determining an expression profile of a set comprising at least one platelet-miRNA from the total RNA isolated.
d. Comparing said expression profile to a reference
e. Identifying if said subject is affected by the disease
f. Optionally administering said affected subject to therapy suitable for said disease
In a still further embodiment, the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2, more preferably said at least one miRNA is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 32, 34, 37, 38, 40, 42, 43, 44, 46, 47, 49, 51, 52.
In a still further embodiment, the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 14, 15, 17, 18, 21, 22, 24, 25, 27, 32, 35.
In a still further embodiment, the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 6, 7, 8, 10, 11, 15, 17, 18, 19, 23.
In a still further embodiment, the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 11 , 13, 16, 20, 21 , 22, 25, 26, 27, 28, 30, 31, 36, 41, 45.
In a still further embodiment, the disease is Multiple Sclerosis and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 6, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 5, 11 , 12, 13, 16, 19, 20, 21 , 23, 24, 25, 26, 28, 29, 31, 32, 37, 39, 50, 55.
In a still further embodiment, the disease is Benign Prostate Hyperblasia (BPH) and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 7, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 16, 17, 18, 19, 21, 37, 40, 41.
In a still further embodiment, the disease is Pancreatitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 8, more preferably said at least one miRNA is selected from the group SEQ ID NO: 12, 23, 26, 27, 29, 33, 35.
In a still further embodiment, the disease is Wilms' Tumor and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 9, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 30, 41, 46, 53.
In a still further embodiment, the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 9, 11, 12, 14, 19, 22, 26.
In a still further embodiment, the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 11 , more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 22, 23, 35, 39, 56.
In a still further embodiment, the disease is Pancreatic Cancer and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 12, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 12, 27, 29, 33.
In a still further embodiment, the disease is Periodontitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 13, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 9, 10, 1 1 , 12, 14, 15, 16, 20, 21, 23, 24, 25, 29, 30, 33, 34, 39.
In a still further embodiment, the disease is COPD and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 14, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 10, 13, 14, 16, 17, 18, 26, 36, 42, 43, 48.
In a still further embodiment, the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 5, 8, 9, 13, 14, 19, 22, 28, 33, 34, 38, 54.
In a still further embodiment, the disease is Ovarian Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 16, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 5, 6, 1 1 , 13, 16, 17, 19, 21, 22, 23, 24, 27, 28, 30, 39, 42, 44, 46, 49.
In a still further embodiment of the second aspect of the present invention, the set comprising at least one platelet-miRNA does not comprise miRNA(s) selected from the group consisting of hsa-miR-126, hsa-miR-197, hsa-miR-223, hsa-miR-21 and hsa-miR-24.
In a third aspect, the invention relates to a method for diagnosing a platelet-related disease in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
It is preferred that in said method the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to 56.
It is preferred that the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a Paxgene-like tube, particularly preferred it is collected in a PAXgene™ Blood RNA tube.
It is preferred that the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells. In a further alternative
embodiment, the expression profile is determined from a whole blood sample in front of a background of white blood cells and red blood cells. In a still further alternative embodiment, the expression profile is determined from a whole blood sample without prior isolation (or extraction) of the platelet fraction
Preferably, the method of the third aspect of the present invention including the aforementioned embodiments comprises the following steps :
a. Providing a whole blood sample of a subject
b. Isolation of the total R A from said whole blood sample
c. Determining an expression profile of a set comprising at least one platelet-miRNA from the total RNA isolated.
d. Comparing said expression profile to a reference
e. Identifying if said subject is affected by the disease
f. Optionally administering said affected subject to therapy suitable for said disease In a still further embodiment, the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2, more preferably said at least one miRNA is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 32, 34, 37, 38, 40, 42, 43, 44, 46, 47, 49, 51, 52.
In a still further embodiment, the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 14, 15, 17, 18, 21, 22, 24, 25, 27, 32, 35.
In a still further embodiment, the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 6, 7, 8, 10, 11, 15, 17, 18, 19, 23.
In a still further embodiment, the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 11 , 13, 16, 20, 21 , 22, 25, 26, 27, 28, 30, 31, 36, 41, 45.
In a still further embodiment, the disease is Multiple Sclerosis and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 6, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 5, 11 , 12, 13, 16, 19, 20, 21, 23, 24, 25, 26, 28, 29, 31, 32, 37, 39, 50, 55.
In a still further embodiment, the disease is Benign Prostate Hyperblasia (BPH) and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 7, more
preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 16, 17, 18, 19, 21, 37, 40, 41.
In a still further embodiment, the disease is Pancreatitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 8, more preferably said at least one miRNA is selected from the group SEQ ID NO: 12, 23, 26, 27, 29, 33, 35.
In a still further embodiment, the disease is Wilms' Tumor and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 9, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 30, 41, 46, 53.
In a still further embodiment, the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 6, 7, 8, 9, 11 , 12, 14, 19, 22, 26.
In a still further embodiment, the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 11, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 22, 23, 35, 39, 56.
In a still further embodiment, the disease is Pancreatic Cancer and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 12, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 12, 27, 29, 33.
In a still further embodiment, the disease is Periodontitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 13, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 9, 10, 1 1 , 12, 14, 15, 16, 20, 21, 23, 24, 25, 29, 30, 33, 34, 39.
In a still further embodiment, the disease is COPD and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 14, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 10, 13, 14, 16, 17, 18, 26, 36, 42, 43, 48.
In a still further embodiment, the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 5, 8, 9, 13, 14, 19, 22, 28, 33, 34, 38, 54.
In a still further embodiment, the disease is Ovarian Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 16, more preferably said at
least one miRNA is selected from the group SEQ ID NO: 1 , 2, 5, 6, 1 1 , 13, 16, 17, 19, 21 , 22,
23, 24, 27, 28, 30, 39, 42, 44, 46, 49.
In a still further embodiment of the third aspect of the present invention, the set comprising at least one platelet-miRNA does not comprise miRNA(s) selected from the group consisting of hsa-miR-126, hsa-miR-197, hsa-miR-223, hsa-miR-21 and hsa-miR-24.
In a fourth aspect, the invention relates to a method for monitoring the progression of a platelet- related disease in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
It is preferred that in said method the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to 56.
It is preferred that the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a Paxgene-like tube, particularly preferred it is collected in a PAXgene™ Blood RNA tube.
It is preferred that the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells. In a further alternative embodiment, the expression profile is determined from a whole blood sample in front of a background of white blood cells and red blood cells. In a still further alternative embodiment, the expression profile is determined from a whole blood sample without prior isolation (or extraction) of the platelet fraction
Preferably, the method of the fourth aspect of the present invention including the aforementioned embodiments comprises the following steps :
a. Providing a whole blood sample of a subject
b. Isolation of the total RNA from said whole blood sample
c. Determining an expression profile of a set comprising at least one platelet-miRNA from the total RNA isolated.
d. Comparing said expression profile to a reference
e. Identifying if said subject is affected by the progression of the disease
f. Optionally administering said affected subject to therapy suitable for said disease
In a still further embodiment, the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2, more preferably said at least
one miRNA is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 32, 34, 37, 38, 40, 42, 43, 44, 46, 47, 49, 51, 52.
In a still further embodiment, the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 14, 15, 17, 18, 21, 22, 24, 25, 27, 32, 35.
In a still further embodiment, the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 6, 7, 8, 10, 11, 15, 17, 18, 19, 23.
In a still further embodiment, the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 11 , 13, 16, 20, 21 , 22, 25, 26, 27, 28, 30, 31, 36, 41, 45.
In a still further embodiment, the disease is Multiple Sclerosis and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 6, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 5, 11 , 12, 13, 16, 19, 20, 21, 23, 24, 25, 26, 28, 29, 31, 32, 37, 39, 50, 55.
In a still further embodiment, the disease is Benign Prostate Hyperblasia (BPH) and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 7, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 16, 17, 18, 19, 21, 37, 40, 41.
In a still further embodiment, the disease is Pancreatitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 8, more preferably said at least one miRNA is selected from the group SEQ ID NO: 12, 23, 26, 27, 29, 33, 35.
In a still further embodiment, the disease is Wilms' Tumor and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 9, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 30, 41, 46, 53.
In a still further embodiment, the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 6, 7, 8, 9, 11 , 12, 14, 19, 22, 26.
In a still further embodiment, the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 11 , more preferably said at
least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 22, 23, 35, 39, 56.
In a still further embodiment, the disease is Pancreatic Cancer and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 12, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 12, 27, 29, 33.
In a still further embodiment, the disease is Periodontitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 13, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 9, 10, 1 1 , 12, 14, 15, 16, 20, 21, 23, 24, 25, 29, 30, 33, 34, 39.
In a still further embodiment, the disease is COPD and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 14, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 10, 13, 14, 16, 17, 18, 26, 36, 42, 43, 48.
In a still further embodiment, the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 5, 8, 9, 13, 14, 19, 22, 28, 33, 34, 38, 54.
In a still further embodiment, the disease is Ovarian Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 16, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 5, 6, 11 , 13, 16, 17, 19, 21 , 22, 23, 24, 27, 28, 30, 39, 42, 44, 46, 49.
In a still further embodiment of the fourth aspect of the present invention, the set comprising at least one platelet-miRNA does not comprise miRNA(s) selected from the group consisting of hsa-miR-126, hsa-miR-197, hsa-miR-223, hsa-miR-21 and hsa-miR-24.
In a fifth aspect, the invention relates to a method for determining the platelet activity in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
It is preferred that in said method the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to 56.
It is preferred that the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a Paxgene-like tube, particularly preferred it is collected in a PAXgene™ Blood RNA tube.
It is preferred that the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells. In a further alternative embodiment, the expression profile is determined from a whole blood sample in front of a background of white blood cells and red blood cells. In a still further alternative embodiment, the expression profile is determined from a whole blood sample without prior isolation (or extraction) of the platelet fraction
Preferably, the method of the fifth aspect of the present invention including the aforementioned embodiments comprises the following steps :
a. Providing a whole blood sample of a subject
b. Isolation of the total RNA from said whole blood sample
c. Determining an expression profile of a set comprising at least one platelet-miRNA from the total RNA isolated.
d. Comparing said expression profile to a reference
e. Identifying if said subject is affected by non-optimal platelet activity
f. Optionally administering said affected subject to therapy suitable to optimize the platelet activity
It is understood that a non-optimal platelet activity may be a platelet activity that is above or below a certain threshold. Hence the platelet activity may be increased or decreased when compared to said reference. Therefore upon administering said subject to therapy, drugs or other therapeutic measures may be applied with the goal to optimize the platelet activity or to bring the platelet-activity to a normal level, which is not above or below a certain threshold.
In a still further embodiment, the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2, more preferably said at least one miRNA is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 32, 34, 37, 38, 40, 42, 43, 44, 46, 47, 49, 51, 52.
In a still further embodiment, the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 14, 15, 17, 18, 21, 22, 24, 25, 27, 32, 35.
In a still further embodiment, the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 6, 7, 8, 10, 11, 15, 17, 18, 19, 23.
In a still further embodiment, the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5, more preferably said at least
one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 7, 8, 9, 1 1, 13, 16, 20, 21, 22, 25, 26, 27, 28, 30, 31, 36, 41, 45.
In a still further embodiment, the disease is Multiple Sclerosis and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 6, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 5, 11 , 12, 13, 16, 19, 20, 21, 23, 24, 25, 26, 28, 29, 31, 32, 37, 39, 50, 55.
In a still further embodiment, the disease is Benign Prostate Hyperblasia (BPH) and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 7, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 16, 17, 18, 19, 21, 37, 40, 41.
In a still further embodiment, the disease is Pancreatitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 8, more preferably said at least one miRNA is selected from the group SEQ ID NO: 12, 23, 26, 27, 29, 33, 35.
In a still further embodiment, the disease is Wilms' Tumor and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 9, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 30, 41, 46, 53.
In a still further embodiment, the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 6, 7, 8, 9, 11 , 12, 14, 19, 22, 26.
In a still further embodiment, the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 1 1 , more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 22, 23, 35, 39, 56.
In a still further embodiment, the disease is Pancreatic Cancer and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 12, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 12, 27, 29, 33.
In a still further embodiment, the disease is Periodontitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 13, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 9, 10, 1 1 , 12, 14, 15, 16, 20, 21, 23, 24, 25, 29, 30, 33, 34, 39.
In a still further embodiment, the disease is COPD and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 14, more preferably said at
least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 10, 13, 14, 16, 17, 18, 26, 36, 42, 43, 48.
In a still further embodiment, the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 5, 8, 9, 13, 14, 19, 22, 28, 33, 34, 38, 54.
In a still further embodiment, the disease is Ovarian Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 16, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 5, 6, 11 , 13, 16, 17, 19, 21 , 22, 23, 24, 27, 28, 30, 39, 42, 44, 46, 49.
In a still further embodiment of the fifth aspect of the present invention, the set comprising at least one platelet-miRNA does not comprise miRNA(s) selected from the group consisting of hsa-miR-126, hsa-miR-197, hsa-miR-223, hsa-miR-21 and hsa-miR-24. In a sixth aspect, the invention relates to a method for monitoring the efficacy of an anti-platelet therapy in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
It is preferred that in said method the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to 56.
It is preferred that the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a Paxgene-like tube, particularly preferred it is collected in a PAXgene™ Blood RNA tube.
It is preferred that the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells. In a further alternative embodiment, the expression profile is determined from a whole blood sample in front of a background of white blood cells and red blood cells. In a still further alternative embodiment, the expression profile is determined from a whole blood sample without prior isolation (or extraction) of the platelet fraction
Preferably, the method of the sixth aspect of the present invention including the aforementioned embodiments comprises the following steps :
a. Providing a whole blood sample of a subject
b. Isolation of the total RNA from said whole blood sample
c. Determining an expression profile of a set comprising at least one platelet-miRNA from the total RNA isolated.
d. Comparing said expression profile to a reference
e. Identifying if said subject is affected by non-optimal efficacy of an anti-platelet therapy
f. Optionally administering said affected subject to therapy suitable to optimize the anti-platelet therapy
It is understood that a non-optimal efficacy of anti-platelet therapy may be an anti-platelet therapy that is above or below a certain threshold. Hence the efficacy of the anti-platelet therapy may be increased or decreased when compared to said reference. Therefore upon administering said subject to therapy, drugs or other therapeutic measures may be applied with the goal to optimize the efficacy of the anti-platelet therapy or to bring the efficacy of the anti-platelet therapy to a normal level, which is not above or below a certain threshold. In a still further embodiment, the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2, more preferably said at least one miRNA is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 27, 28, 30, 31, 32, 34, 37, 38, 40, 42, 43, 44, 46, 47, 49, 51, 52.
In a still further embodiment, the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 3, 4, 7, 8, 9, 10, 14, 15, 17, 18, 21, 22, 24, 25, 27, 32, 35.
In a still further embodiment, the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 6, 7, 8, 10, 11, 15, 17, 18, 19, 23.
In a still further embodiment, the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 3, 4, 5, 6, 7, 8, 9, 1 1, 13, 16, 20, 21, 22, 25, 26, 27, 28, 30, 31, 36, 41, 45.
In a still further embodiment, the disease is Multiple Sclerosis and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 6, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 5, 11 , 12, 13, 16, 19, 20, 21, 23, 24, 25, 26, 28, 29, 31, 32, 37, 39, 50, 55.
In a still further embodiment, the disease is Benign Prostate Hyperblasia (BPH) and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 7, more
preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 16, 17, 18, 19, 21, 37, 40, 41.
In a still further embodiment, the disease is Pancreatitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 8, more preferably said at least one miRNA is selected from the group SEQ ID NO: 12, 23, 26, 27, 29, 33, 35.
In a still further embodiment, the disease is Wilms' Tumor and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 9, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 30, 41, 46, 53.
In a still further embodiment, the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 2, 3, 4, 6, 7, 8, 9, 11 , 12, 14, 19, 22, 26.
In a still further embodiment, the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 1 1 , more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 17, 18, 20, 22, 23, 35, 39, 56.
In a still further embodiment, the disease is Pancreatic Cancer and said at least one platelet- miRNA comprised in the set is selected from the miRNAs listed in Figure 12, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 12, 27, 29, 33.
In a still further embodiment, the disease is Periodontitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 13, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1 , 3, 4, 5, 6, 9, 10, 1 1 , 12, 14, 15, 16, 20, 21, 23, 24, 25, 29, 30, 33, 34, 39.
In a still further embodiment, the disease is COPD and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 14, more preferably said at least one miRNA is selected from the group SEQ ID NO: 1, 2, 3, 4, 6, 7, 8, 10, 13, 14, 16, 17, 18, 26, 36, 42, 43, 48.
In a still further embodiment, the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15, more preferably said at least one miRNA is selected from the group SEQ ID NO: 2, 5, 8, 9, 13, 14, 19, 22, 28, 33, 34, 38, 54.
In a still further embodiment, the disease is Ovarian Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 16, more preferably said at
least one miRNA is selected from the group SEQ ID NO: 1 , 2, 5, 6, 11 , 13, 16, 17, 19, 21 , 22, 23, 24, 27, 28, 30, 39, 42, 44, 46, 49.
In a still further embodiment of the sixth aspect of the present invention, the set comprising at least one platelet-miRNA does not comprise miRNA(s) selected from the group consisting of hsa-miR-126, hsa-miR-197, hsa-miR-223, hsa-miR-21 and hsa-miR-24.
In a seventh aspect, the invention relates to a method for determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
It is preferred that in said method the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to 56.
It is preferred that the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a Paxgene-like tube, particularly preferred it is collected in a PAXgene™ Blood RNA tube.
It is preferred that the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells. In a further alternative embodiment, the expression profile is determined from a whole blood sample in front of a background of white blood cells and red blood cells. In a still further alternative embodiment, the expression profile is determined from a whole blood sample without prior isolation (or extraction) of the platelet fraction
Preferably, the method of the seventh aspect of the present invention including the aforementioned embodiments comprises the following steps :
a. Providing a whole blood sample of a subject
b. Isolation of the total RNA from said whole blood sample
c. Determining an expression profile of a set comprising at least one platelet-miRNA from the total RNA isolated.
In a further embodiment the determining of an expression profile of a set comprising at least one platelet-miRNA (selected from SEQ ID NO: 1 to 56) from the total RNA isolated according to the first aspect (diagnosing a platelet-related component of a disease), to the second aspect (diagnosing a disease), to the third aspect (diagnosing a platelet-related disease), to the fourth aspect (monitoring the progression of a disease), to the fifth aspect (determining the platelet activity of a disease), to the sixth aspect (monitoring the efficacy of an anti-platelet-therapy) or
to the seventh aspect (determining an expression profile of a set comprising at least on platelet- miRNA) of the invention comprises the steps :
(a) reverse-transcribing the at least one platelet-miRNA comprised in the total RNA isolated (from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells) into non-naturally occurring cDNA
(b) optionally amplifying the cDNA of step (a)
(c) quantifying the optionally amplified cDNA, thereby determining the expression profile of said miRNAs
Herein it is preferred that miRNA-specific or universal reverse transcription DNA-primers are used for reverse transcription in step (a). Examples of miRNA-specific reverse transcription primers are listed in column B of Figure 17, examples of universal reverse transcription primers are 01igo-d(T)-Primers or RT primers listed in column C of Figure 20. It is further preferred that miRNA-specific forward primer and universal reverse primer or alternatively universal forward and universal reverse primer are used for optionally amplifying the cDNA in step (b). Examples of miRNA-specific forward primer and universal reverse primer are listed in column C and D of Figure 17, examples of universal forward and universal reverse primer are listed in column D and E of Figure 20. Further, it is preferred that miRNA-specific forward primer and universal reverse primer or alternatively miRNA-specific forward and partially universal reverse primer are used for quantifying the optionally amplified cDNA in step (c). It is preferred that the quantifying in step (c) is performed by real-time PCR, nucleic acid hybridization (e.g. microarray) or sequencing (e.g. next generation sequencing) techniques. It is preferred that the quantifying in step (c) by real-time PCR is utilizing dual-labeled hydrolysis probes that make use of the 5 '-3 ' exonuclease activity of polymerase (e.g. Taqman-probes) or DNA-intercalating dyes (e.g. SYBRgreen). Examples of miRNA-specific forward primer and universal reverse primer are listed in column C and D of Figure 17, examples of miRNA-specific forward and partially universal reverse primer are listed in column B and C of Figure 18; examples of dual-labeled hydrolysis probes are listed in column E of Figure 17.
In a still further embodiment the determining of an expression profile of a set comprising at least one platelet-miRNA (selected from SEQ ID NO: 1 to 56) from the total RNA isolated according to the first aspect (diagnosing a platelet-related component of a disease), to the second aspect (diagnosing a disease), to the third aspect (diagnosing a platelet-related disease), to the fourth aspect (monitoring the progression of a disease), to the fifth aspect (determining the platelet activity of a disease), to the sixth aspect (monitoring the efficacy of an anti-platelet-therapy) or to the seventh aspect (determining an expression profile of a set comprising at least on platelet- miRNA) of the invention comprises the steps :
(a) adding a DN A- fragment to the 3 '-end of the at least one platelet-miRNA comprised in the total RNA isolated (from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells), thereby forming non-naturally occurring RNA-DNA hybrids
(b) optionally reverse-transcribing said RNA-DNA hybrids to cDNA
(c) quantifying the optionally reverse-transcribed RNA-DNA hybrids, thereby determining the expression profile of said miRNAs
Herein it is preferred that DNA- fragments of 1 to 150 nucleotides in length (preferably of 1 to 100 nt, more preferably of 1 to 50 nt, even more preferably of 1 to 30 nt in length) are added in step (a) to the 3 '-end of the miRNAs by ligation or by polymerase-based elongation. Examples of said DNA-fragments are listed in Figure 19 or column A of Figure 20.
It is preferred that (miRNA-specific or) universal reverse transcription DNA-primers are used for reverse transcription in step (b). Further, it is preferred that miRNA-specific forward primer and universal reverse primer or alternatively miRNA-specific forward and partially universal reverse primer are used for quantifying the optionally amplified cDNA in step (c). Examples of miRNA- specific reverse transcription primers are listed in column B of Figure 17, examples of universal reverse transcription primers are oligo-d(T)-Primers or RT primers listed in column C of Figure 20. It is preferred that the quantifying in step (c) is performed by real-time PCR, nucleic acid hybridization or sequencing (e.g. next generation sequencing) techniques. It is preferred that the quantifying in step (c) by real-time PCR is utilizing dual-labeled hydrolysis probes that make use of the 5 '-3 ' exonuclease activity of polymerase (e.g. Taqman-probes) or DNA-intercalating dyes (e.g. SYBRgreen).
In a still further embodiment the determining of an expression profile of a set comprising at least one platelet-miRNA (selected from SEQ ID NO: 1 to 56) from the total RNA isolated according to the first aspect (diagnosing a platelet-related component of a disease), to the second aspect (diagnosing a disease), to the third aspect (diagnosing a platelet-related disease), to the fourth aspect (monitoring the progression of a disease), to the fifth aspect (determining the platelet activity of a disease), to the sixth aspect (monitoring the efficacy of an anti-platelet-therapy) or to the seventh aspect (determining an expression profile of a set comprising at least on platelet- miRNA) of the invention comprises the steps :
(a) adding a RNA- fragment to the 3 '-end of the at least one platelet-miRNA comprised in the total RNA isolated (from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells), thereby forming non-naturally occurring RNA-RNA hybrids
(b) optionally reverse-transcribing said RNA-RNA hybrids to cDNA
(c) quantifying the optionally reverse-transcribed RNA-RNA hybrids, thereby determining the expression profile of said miRNAs
Herein it is preferred that RNA- fragments of 1 to 150 nucleotides in length (preferably of 1 to 100 nt, more preferably of 1 to 50 nt, even more preferably of 1 to 30 nt in length) are added in step (a) to the 3 '-end of the miRNAs preferably by poly(A)-tailing reaction. The reverse- transcription of step (b) is preferably with universal RT -primers, e.g. oligo-d(T)-primers. The quantifying in step (c) is preferably utilizing miRNA-specific forward and partially universal reverse primer, e.g. miRNA-specific forward and partially universal reverse primer as listed in column B and C of Figure 18.
In an eighth aspect, the invention relates the use of a whole blood collection tube in the method according to any of the first, second, third, fourth, fifth, sixth of seventh aspect of the present invention including all its embodiments.
It is preferred that a whole blood collection tube in the use according to the eight aspect of the invention contains an additive for RNA stabilization, preferably optionally contians an additive for stabilization of intracellular RNA, more preferably in said use the whole blood collection tube is a PAXgene™ Blood RNA tube. Preferably, said additive for RNA-stabilization is selected from the group consisting of catrimox-14, tetradecyltrimethylammonium oxalate, RNA- later, RNAsin or RNAretain.
It is particularly preferred that a PAXgene™ Blood RNA tube is employed in the use according to the eight aspect of the invention for determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells. In a ninth aspect, the invention relates to a kit for use in the method according to any of the first, second, third, fourth, fifth, sixth of seventh aspect of the present invention including all of its embodiments.
Said kit for use in the method according to any of the first, second, third, fourth, fifth, sixth of seventh aspect of the present invention comprises :
a) means for determining an expression profile of a set comprising at least one platelet-miRNA
b) a reference derived from at least one reference expression profile
c) optionally a data carrier
d) optionally a whole blood collection tube
wherein the expression profile and the reference expression profile are obtained from said set of at least one platelet-miR As selected from the group consisting SEQ ID NO: 1 to 56 and wherein the expression profile and the reference expression profile are determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
In a preferred embodiment, the reference may be contained in the data carrier of the kit. In a further preferred embodiment the reference may be a reference sample and/or a reference standard that is included in the kit and which is employed when performing the kit, e.g. in the determining of the expression profile. The kit comprises a data carrier. Preferably the data carrier is an electronic or a non-electronic data carrier, more preferably it is an electronic data carrier, such as a storage medium.
It is preferred that the data carrier comprised in the kit comprises a guide for use of the kit in the method according to any of the first, second, third, fourth, fifth, sixth of seventh aspect of the present invention. This guide may include instructions for the doctor and/or the diagnostic laboratory that are involved in the method according to any of the first, second, third, fourth, fifth, sixth of seventh aspect of the present invention. The guide may include a reference according to the present invention.
It is preferred that the data carrier further comprises tools for analysis and evaluation of the determined expression profile(s). These tools may be any tools to assist the doctor and/or the diagnostic laboratory in the method according to any of the first, second, third, fourth, fifth, sixth of seventh aspect of the present invention. Preferably, these tools are software-tools that assist in analysis of the determined expression profile(s) and/or assist in the subsequently diagnosis. The tools for analysis and evaluation may include a reference according to the present invention. The kit optionally comprises whole bood collection tubes, which are preferably selected from group consisting of EDTA- , Na-citrate-, ACD-, Heparin-, PAXgene™ Blood RNA-, Tempus Blood RNA-tubes and optionally contains an additive for stabilizing the RNA-fraction.
In summary, the present invention is composed of the following items :
1. Method for diagnosing a platelet-related or platelet-activated component of a disease by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
Method for diagnosing a disease by determining an expression profile of a set comprising at least one platelet-miR A from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
Method for diagnosing a platelet-related disease in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
Method for monitoring the progression of a platelet-related disease in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
Method for determining the platelet activity in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
Method for monitoring the efficacy of an anti-platelet therapy in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
The method according to any of the items 1 to 6, comprising the steps : a. Providing a whole blood sample of a subject b. Isolation of the total RNA from said whole blood sample c. Determining an expression profile of a set comprising at least one platelet- miRNA from the total RNA isolated
d, Comparing said expression profile to a reference e. Identifying if said subject is affected by one of the disease conditions (detailed in items 1 to 6 f Optionally administering said affected subject to therapy of said disease condition The method according to item 7, wherein the disease condition is selected from the group consisting of platelet-related or platelet-activated component of a disease, disease, platelet- related disease, progression of a platelet-related disease, platelet activity or efficacy of an anti-platelet therapy. Method for determining an expression profile of a set comprising at least one platelet- miR A from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells. The method according to any of the items 1 to 9, wherein the platelet-miRNAs are selected from the group consisting of SEQ ID NO: 1 to 56. The method according to any of the items 1 to 10, wherein the whole blood sample is collected in a whole blood collection tube, optionally containing an additive for RNA stabilization, preferably optionally containing an additive for stabilization of intracellular RNA, more preferably the whole blood is collected in a PAXgene™ Blood RNA tube.
The method according to item 11, wherein the additive for RNA-stabilization is selected from the group consisting of catrimox-14, tetradecyltrimethylammonium oxalate, RNA- later, RNAsin or RNAretain.
Use of a whole blood collection tube in the method according to any of the items 1 to 12 wherein the expression profile is determined from the total RNA isolated from the whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
The use according to item 13, wherein the whole blood collection tube is a PAXgene Blood RNA tube.
The method or use according to any of the items 1 to 14, wherein the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2. The method or use according to any of the items 1 to 14 , wherein the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3. The method or use according to any of the items 1 to 14, wherein the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4. The method or use according to any of the items 1 to 14, wherein the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5. The method or use according to any of the items 1 to 14, wherein the disease is Multiple Sclerosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 6.
The method or use according to any of the items 1 to 14, wherein the disease is Benign Prostate Hyperblasia (BPH) and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 7. The method or use according to any of the items 1 to 14, wherein the disease is Pancreatitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 8. The method or use according to any of the items 1 to 14, wherein the disease is Wilm's Tumor and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 9. The method or use according to any of the items 1 to 14, wherein the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10.
The method or use according to any of the items 1 to 14, wherein the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miR As listed in Figure 11. The method or use according to any of the items 1 to 14, wherein the disease is Pancreatic Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 12. The method or use according to any of the items 1 to 14, wherein the disease is Periodontitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 13. The method or use according to any of the items 1 to 14, wherein the disease is COPD and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 14. The method or use according to any of the items 1 to 14, wherein the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15. The method or use according to any of the items 1 to 14, wherein the disease is Ovarian Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 16. A kit for use in the method according to any of the items 1 to 12 or 15 to 29, comprising : a) means for determining an expression profile of a set comprising at least one platelet-miRNA
b) a reference derived from at least one reference expression profile
c) optionally a data carrier
d) optionally a whole blood collection tube as detailed in items 11 or 12
wherein the expression profile and the reference expression profile are obtained from said at least one platelet-miRNAs selected from the group consisting SEQ ID NO: 1 to 56 and wherein the expression profile and the reference expression profile are
determined from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells. The kit according to item 30, wherein the whole blood collection tube is a PAXgene Blood RNA tube.
Use of a PAXgene™ Blood RNA tube for determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from the total RNA isolated from the whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
The use according to item 32, wherein the platelet-miRNAs are selected from the group consisting of SEQ ID NO: 1 to 56.
The method according to any of the items 1 to 12 or 15 to 29, wherein the determining of an expression profile of a set comprising at least one platelet-miRNA comprises the steps:
(a) reverse-transcribing the at least one platelet-miRNA comprised in the total RNA isolated into non-naturally occurring cDNA
(b) optionally amplifying the cDNA of step (a)
(c) quantifying the optionally amplified cDNA, thereby determining the expression profile of said at least one platelet-miRNA
The method according to item 34, wherein miRNA-specific or universal reverse transcription DNA-primers are used for reverse transcription in step (a).
The method according to any of the items 34 to 35, wherein miRNA-specific forward primer and universal reverse primer or miRNA-specific forward and partially universal reverse primer are used for quantifying the optionally amplified cDNA in step (c).
The method according to any of the items 34 to 37, wherein miRNA-specific forward primer and universal reverse primer or miRNA-specific forward and partially universal reverse primer are used for optionally amplifying the cDNA in step (b).
38 The method according to any of the items 34 to 38 wherein that the quantifying in step is performed by real-time PCR, nucleic acid hybridization or sequencing techniques.
The method according to any of the items 1 to 12 or 15 to 29, wherein the determining of an expression profile of a set comprising at least one platelet-miRNA comprises the steps:
(a) adding a DNA- fragment to the 3 '-end of the at least one platelet-miRNA comprised in the total RNA isolated, thereby forming non-naturally occurring RNA-DNA hybrids
(b) optionally reverse-transcribing said RNA-DNA hybrids to cDNA
(c) quantifying the optionally reverse-transcribed RNA-DNA hybrids, thereby determining the expression profile of said at least one platelet-miRNA
The method according to item 39, wherein DNA-fragments of 1 to 150 nucleotides in length are added to the 3 '-end of the miRNAs by ligation or by polymerase-based elongation.
The method according to any of the items 39 to 40, wherein universal reverse transcription DNA-primers are used for reverse transcription in step (b).
The method according to any of the items 39 to 41, wherein miRNA-specific forward primer and universal reverse primer or miRNA-specific forward and partially universal reverse primer are used for quantifying the optionally amplified cDNA in step (c).
The method according to any of the items 39 to 42, wherein that the quantifying in step (c) is performed by real-time PCR, nucleic acid hybridization or sequencing techniques.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 : Overview of the platelet-miRNAs (with SEQ ID NO: 1 to 56) well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention. With "SEQ ID NO:" = sequence identification number,
"miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Sequence" =
(5'-3')-sequence of the miRNA.
Figure 2: SARCOIDOSIS : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet- related disease, for determining the platelet-activity and/or for monitoring the efficacy of antiplatelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Sarcoidosis. With "SEQ ID NO:" = sequence identification number, "miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Healthy Control median gl" = median intensity (expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "Sarcoidosis median g2" = " = median intensity (expression level) obtained from microarray analysis for Sarcoidosis subjects in counts/sec; "Fold Change (gl/g2)" = expression fold-change by calculating the ration of the values gl/g2 from "Healthy Control median gl" and "Sarcoidosis median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false- discovery rate by applying Benjamini-Hochberg-adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false-discovery rate by applying Benjamini- Hochberg-adjustment; "AUC" = area under the curve- value of ROC-curve statistical model.
Figure 3 : RENAL CANCER : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Renal Cancer. With "SEQ ID NO:" = sequence identification number, "miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Healthy Control median gl" = median intensity (expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "Renal Cancer median g2" = " = median intensity (expression level) obtained from microarray analysis for Renal Cancer subjects in counts/sec; "Fold Change (gl/g2)" = expression fold-
change by calculating the ration of the values gl/g2 from "Healthy Control median gl " and "Renal Cancer median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "AUC" = area under the curve-value of ROC-curve statistical model.
Figure 4: COLON CANCER : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Colon Cancer. With "SEQ ID NO :" = sequence identification number, "miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Healthy Control median gl" = median intensity (expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "Colon Cancer median g2" = " = median intensity (expression level) obtained from microarray analysis for Colon Cancer subjects in counts/sec; "Fold Change (gl/g2)" = expression fold- change by calculating the ration of the values gl/g2 from "Healthy Control median gl " and "Colon Cancer median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "AUC" = area under the curve-value of ROC-curve statistical model.
Figure 5: PSORIASIS : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet- related disease, for determining the platelet-activity and/or for monitoring the efficacy of antiplatelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Psoriasis. With "SEQ ID NO:" =
sequence identification number, "miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Healthy Control median gl" = median intensity (expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "Psoriasis median g2" = " = median intensity (expression level) obtained from microarray analysis for Psoriasis subjects in counts/sec; "Fold Change (gl/g2)" = expression fold-change by calculating the ration of the values gl/g2 from "Healthy Control median gl" and "Psoriasis median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "AUC" = area under the curve-value of ROC-curve statistical model.
Figure 6: MULTIPLE SCLEROSIS : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Multiple Sclerosis. With "SEQ ID NO:" = sequence identification number, "miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Healthy Control median gl" = median intensity (expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "Multiple Sclerosis median g2" = " = median intensity (expression level) obtained from microarray analysis for Multiple Sclerosis subjects in counts/sec; "Fold Change (gl/g2)" = expression fold-change by calculating the ration of the values gl/g2 from "Healthy Control median gl" and "Multiple Sclerosis median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false-discovery rate by applying Benjamini-Hochberg- adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "AUC" = area under the curve-value of ROC-curve statistical model.
Figure 7: BENIGN PROSTATIC HYPERBLASIA (BPH) : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for
monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Benign Prostatic Hyperblasia (BPH). With "SEQ ID NO:" = sequence identification number, "miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Healthy Control median gl" = median intensity (expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "Benign Prostatic Hyperblasia (BPH) median g2" = " = median intensity (expression level) obtained from microarray analysis for Benign Prostatic Hyperblasia (BPH) subjects in counts/sec; "Fold Change (gl/g2)" = expression fold-change by calculating the ration of the values gl/g2 from "Healthy Control median gl" and "Benign Prostatic Hyperblasia (BPH) median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false- discovery rate by applying Benjamini-Hochberg-adjustment; "AUC" = area under the curve- value of ROC-curve statistical model.
Figure 8 : PANCREATITIS : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Pancreatitis. With "SEQ ID NO :" = sequence identification number, "miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Healthy Control median gl" = median intensity (expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "Pancreatitis median g2" = " = median intensity (expression level) obtained from microarray analysis for Pancreatitis subjects in counts/sec; "Fold Change (gl/g2)" = expression fold-change by calculating the ration of the values gl/g2 from "Healthy Control median gl" and "Pancreatitis median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false- discovery rate by applying Benjamini-Hochberg-adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false-discovery rate by applying Benjamini- Hochberg-adjustment; "AUC" = area under the curve-value of ROC-curve statistical model.
Figure 9: WILMS' TUMOR : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Wilms' Tumor. With "SEQ ID NO:" = sequence identification number, "miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Healthy Control median gl" = median intensity (expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "Wilms' Tumor median g2" = " = median intensity (expression level) obtained from microarray analysis for Wilms' Tumor subjects in counts/sec; "Fold Change (gl/g2)" = expression fold- change by calculating the ration of the values gl/g2 from "Healthy Control median gl " and "Wilms' Tumor median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "AUC" = area under the curve-value of ROC-curve statistical model.
Figure 10: PROSTATE CANCER : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Prostate Cancer. With "SEQ ID NO :" = sequence identification number, "miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Healthy Control median gl" = median intensity (expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "Prostate Cancer median g2" = " = median intensity (expression level) obtained from microarray analysis for Prostate Cancer subjects in counts/sec; "Fold Change (gl/g2)" = expression fold- change by calculating the ration of the values gl/g2 from "Healthy Control median gl " and
"Prostate Cancer median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "AUC" = area under the curve-value of ROC-curve statistical model.
Figure 1 1 : LUNG CANCER : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Lung Cancer. With "SEQ ID NO :" = sequence identification number, "miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Healthy Control median gl" = median intensity (expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "Lung Cancer median g2" = " = median intensity (expression level) obtained from microarray analysis for Lung Cancer subjects in counts/sec; "Fold Change (gl/g2)" = expression fold- change by calculating the ration of the values gl/g2 from "Healthy Control median gl" and "Lung Cancer median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "AUC" = area under the curve-value of ROC-curve statistical model.
Figure 12: PANCREATIC CANCER : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Pancreatic Cancer. With "SEQ ID NO:" = sequence identification number, "miRNA" = identifier of the miRNA
according to miRBase (www.mirbase.org), "Healthy Control median gl " = median intensity
(expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "Pancreatic Cancer median g2" = " = median intensity (expression level) obtained from microarray analysis for Pancreatic Cancer subjects in counts/sec; "Fold Change (gl/g2)" = expression fold-change by calculating the ration of the values gl/g2 from "Healthy Control median gl" and "Pancreatic Cancer median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false-discovery rate by applying Benjamini-Hochberg- adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "AUC" = area under the curve-value of ROC-curve statistical model.
Figure 13 : PERIODONTITIS : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Periodontitis. With "SEQ ID NO:" = sequence identification number, "miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Healthy Control median gl" = median intensity (expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "Periodontitis median g2" = " = median intensity (expression level) obtained from microarray analysis for Periodontitis subjects in counts/sec; "Fold Change (gl/g2)" = expression fold- change by calculating the ration of the values gl/g2 from "Healthy Control median gl " and "Periodontitis median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "AUC" = area under the curve-value of ROC-curve statistical model.
Figure 14: COPD (Chronic obstructive pulmonary disease): Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for
monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is COPD. With "SEQ ID NO:" = sequence identification number, "miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Healthy Control median gl " = median intensity (expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "COPD median g2" = " = median intensity (expression level) obtained from microarray analysis for COPD subjects in counts/sec; "Fold Change (gl/g2)" = expression fold- change by calculating the ration of the values gl/g2 from "Healthy Control median gl " and "COPD median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "AUC" = area under the curve-value of ROC-curve statistical model.
Figure 15: MELANOMA : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet- related disease, for determining the platelet-activity and/or for monitoring the efficacy of antiplatelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Melanoma. With "SEQ ID NO:" = sequence identification number, "miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Healthy Control median gl" = median intensity (expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "Melanoma median g2" = " = median intensity (expression level) obtained from microarray analysis for Melanoma subjects in counts/sec; "Fold Change (gl/g2)" = expression fold-change by calculating the ration of the values gl/g2 from "Healthy Control median gl" and "Melanoma median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "AUC" = area under the curve-value of ROC-curve statistical model.
Figure 16: OVARIAN CANCER : Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated inflammatory component of a disease, for diagnosing a disease, for diagnosing a platelet-related disease, for monitoring the progression of a platelet-related disease, for determining the platelet-activity and/or for monitoring the efficacy of anti-platelet therapy and for determining an expression profile according to the present invention, wherein the expression profile of the platelet-miRNAs is determined from a whole blood sample collected in a PAXgene™ Blood RNA tube without prior depletion or removal of the white blood cells and/or the red blood cells and wherein the disease is Ovarian Cancer. With "SEQ ID NO :" = sequence identification number, "miRNA" = identifier of the miRNA according to miRBase (www.mirbase.org), "Healthy Control median gl" = median intensity (expression level) obtained from microarray analysis for healthy control subjects in counts/sec; "Ovarian Cancer median g2" = " = median intensity (expression level) obtained from microarray analysis for Ovarian Cancer subjects in counts/sec; "Fold Change (gl/g2)" = expression fold- change by calculating the ration of the values gl/g2 from "Healthy Control median gl " and "Ovarian Cancer median g2"; "ttest adjpval" = adjusted p-value obtained when applying t-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "limma adjpval" = adjusted p-value obtained when applying limma-test and reducing false-discovery rate by applying Benjamini-Hochberg-adjustment; "AUC" = area under the curve-value of ROC-curve statistical model.
Figure 17 : miRNA-specific DNA-primers (column B) used for reverse transcription (RT) of at least one platelet-miRNA with SEQ ID NO: 1 to 56 to non-naturally occurring cDNA; miRNA- specific forward and universal reverse primers (column C, D) for quantification and optionally amplification of at least one platelet-miRNA with SEQ ID NO: 1 to 56 employed for determining of an expression profile of a set comprising at least one platelet-miRNA; dual- labeled hydrolysis probes (Taqman-probes, column E) utilized for quantifying of at least one platelet-miRNA with SEQ ID NO: 1 to 56 by real-time PCR .
Figure 18 : miRNA-specific forward primer (column B) and partially universal reverse primer (column C) for quantification and optionally amplification of cDNA-transcripts of at least one platelet-miRNA with SEQ ID NO: 1 to 56 employed for determining of an expression profile of a set comprising at least one platelet-miRNA.
Figure 19 : DNA- fragments added to the 3 '-end of at least one platelet-miRNA with SEQ ID NO: 1 to 56 employed for determining of an expression profile of a set comprising at least one platelet-miRNA, thereby forming non-naturally occurring RNA-DNA hybrids. Figure 20 :Adapters, RT -primers and PCR-primers utilized for next generation sequencing (Illumina small RNA-seq) of the platelet-miRNAs with SEQ ID NO: 1 to 56 employed for determining of an expression profile of a set comprising at least one platelet-miRNA : universal 3' RNA Adapters (column A) ligated to the 3 '-end of the platelet-miRNAs with SEQ ID NO: 1 to 56; universal 5' RNA Adapter (column B) ligated to the 5 '-end of the at least one platelet- miRNA with SEQ ID NO: 1 to 56; universal reverse transcription (RT)-Primers (column C) for reverse-transcribing the 3'- and 5 '-adapter ligated miRNAs into (non-naturally occurring) cDNA; Small RNA PCR Primer l=universal forward (column D) and Small RNA PCR Primer 2=universal reverse (PCR) primers (column E) for amplifying the 3'- and 5 '-adapter ligated and reverse-transcribed cDNAs of at least one platelet-miRNA with SEQ ID NO: 1 to 56.
EXAMPLES
The Examples are designed in order to further illustrate the present invention and serve a better understanding. They are not to be construed as limiting the scope of the invention in any way.
EXAMPLE 1 : Preparation of total RNA for determination of platelet-miRNAs from whole blood samples without prior depletion or removal of the white blood cells and/or the red blood cells
Blood of patients and healthy controls was drawn in PAXgene™ Blood RNA tubes (PreAnalytiX GmbH, Hombrechtikon, Switzerland). For each blood donor, 2.5 ml of peripheral whole blood was collected. Herein, the blood cell pellet (the intra-cellular blood fraction comprising red blood cells, white blood cells and platelets) was collected at the bottom of the tube by centrifugation. For further processing the blood cell pellet was used, while the supernatant (including the extra-cellular blood fraction) was discarded. Total RNA, including the small RNA (miRNA- fraction) was isolated from the pelleted blood cells using the miRNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and the resulting RNA was stored at -70° C before use in expression profiling experiments.
The healthy control and the patient samples for investigation of Sarcoidosis, Renal Cancer, Colon Cancer, Psoriasis, Multiple Sclerosis, BPH, Pancreatitis, Wilms Tumor, Prostate Cancer,
Lung Cancer, Pancreatic Cancer, Periodontitis, COPD, Melanoma, Ovarian Cancer were prepared as detailed in Example 1.
EXAMPLE 2 : Microarray-based determination of expression profiles
The RNA-samples were analyzed employing microarray hybridization on the Geniom Realtime Analyzer (febit biomed GmbH, Heidelberg, Germany) using the Geniom Biochip miRNA homo sapiens. Each microfluidic microarray contains complementary dna-probes of 866 miRNAs and miRNA* (each represented by 7 replicates) as annotated in the Sanger miRBase 12.0. Sample labeling with biotin has been carried out by enzymatic on-chip labeling of miRNAs employing the MPEA-assay (Vorwerk et.al. N Biotechnol. 2008, 25(2-3): 142-9). Following hybridization for 16 hours at 42°C the biochip was washed automatically and a program for signal enhancement was processed with the Geniom Realtime Analyzer. The resulting detection pictures were evaluated using the Geniom Wizard Software. For each array, the median signal intensity was extracted from the raw data file such that for each miRNA seven intensity values have been calculated corresponding to each replicate copy of miRBase on the array. Following background correction, the seven replicate intensity values of each miRNA were summarized by their median value. To normalize the data across different arrays, quantile normalization was applied and all further analyses were carried out using the normalized and background subtracted intensity values.
The data for investigation of Sarcoidosis, Renal Cancer, Colon Cancer, Psoriasis, Multiple Sclerosis, BPH, Pancreatitis, Wilms Tumor, Prostate Cancer, Lung Cancer, Pancreatic Cancer, Periodontitis, COPD, Melanoma, Ovarian Cancer were obtained by microarray analysis as detailed in Example 2. EXAMPLE 3 : Statistical Analysis
After having verified the normal distribution of the measured data, a parametric t-test (unpaired, two-tailed) was carried out for each miRNA separately, to detect miRNAs that show a different behavior in different groups of blood donors. The resulting p-values were adjusted for multiple testing by Benjamini-Hochberg adjustment (=ttestadjpval). Furthermore, we applied the limma- test for each miRNA separately and corrected according to Benjamini-Hochberg (= limma adjpval). Additionally, we applied receiver operating characteristics and calculated the "Area under the Curve"-value (=AUC). The ttest-, limma-test- and AUC-values allow to judge on the statistical significance for each miRNA to be differential expressed between group 1 (gl = Healthy Control subjects) and group 2 (g2=disease subjects).
EXAMPLE 4 : Data Filtering
For each dataset (e.g. Healthy Control vs. Sarcoidosis, Healthy Control vs. Renal Cancer , Healthy Control vs. Colon Cancer etc.; see Figures 2-20, Example 5) strict filtering rules were applied to result finally in the platelet-miRNAs with SEQ IDNO: 1 to 56. In each dataset all miRNAs were discarded that had (a) low expression levels as shown by a median intensity gl or g2 (expression level from microarray analysis) below 100 counts/sec or that had (b) low differential expression as shown by an expression fold change ratio gl/g2 in between 0.66 and 1.5 (translating to a fold change factor of 1.5). By manual curating of the data a few exceptions to the filtering rules outlined above were allowed, when the g2 intensity (disease) was below the 100 count/sec threshold, but the gl intensity (healthy control) showed strong expression, resulting in a strong down-regulation in the disease.
Next, the remaining miRNA-biomarker candidates were aligned to a list of selected platelet- miRNAs described to be highly expressed in platelets (PMID 21415270, 22371016, 23323973), wherein the list of highly expressed miRNAs contained the 30% highest expressed platelet- miRNAs.
EXAMPLE 5 : Patient Characteristics
Claims
1. Method for diagnosing a platelet-related component of a disease by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior removal of the white blood cells and/or the red blood cells.
2. Method for diagnosing a platelet-related disease in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior removal of the white blood cells and/or the red blood cells.
3. Method for monitoring the progression of a platelet-related disease in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior removal of the white blood cells and/or the red blood cells.
4. The method according to any of the claims 1 to 3, comprising the steps : a. Providing a whole blood sample of a subject
b. Isolation of the total RNA from said whole blood sample
c. Determining an expression profile of a set comprising at least one platelet- miRNA from the total RNA isolated
d. Comparing said expression profile to a reference
e. Identifying if said subject is affected by the disease
f. Optionally administering said affected subject to therapy
5. Method for determining an expression profile of a set comprising at least one platelet- miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a whole blood sample without prior removal of the white blood cells and/or the red blood cells.
6. The method according to claim 5, comprising the steps
a. Providing a whole blood sample of a subject
b. Isolation of the total RNA from said whole blood sample
c. Determining an expression profile of a set comprising at least one platelet- miRNA from the total RNA isolated
7. The method according to any of the claims 1 to 6, wherein the platelet-miRNAs are selected from the group consisting of SEQ ID NO: 1 to 56.
8. The method according to any of the claims 1 to 7, wherein the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a PAXgene™ Blood RNA tube.
9. Use of a whole blood collection tube, preferably a PAXgene™ Blood RNA tube in the method according to any of the claims 1 to 8 wherein the expression profile of a set comprising at least one platelet-miRNA is determined from the total RNA isolated from the whole blood sample of a subject without prior removal of the white blood cells and/or the red blood cells.
10. A kit for use in the method according to any of the claims 1 to 8, comprising :
a) means for determining an expression profile of a set comprising at least one platelet-miRNA
b) a reference derived from at least one reference expression profile
c) optionally a data carrier
d) optionally a whole blood collection tube wherein the expression profile and the reference expression profile are obtained from said set of at least one platelet-miRNA selected from the group consisting SEQ ID NO: 1 to 56 and wherein the expression profile and the reference expression profile are determined from a whole blood sample without prior removal of the white blood cells and/or the red blood cells.
11. The method or use according to any of the claims 1 to 8, wherein the disease is Colon Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 4.
12. The method or use according to any of the claims 1 to 8, wherein the disease is Sarcoidosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 2.
13. The method or use according to any of the claims 1 to 8, wherein the disease is Psoriasis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 5.
14. The method or use according to any of the claims 1 to 8, wherein the disease is Multiple Sclerosis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 6.
15. The method or use according to any of the claims 1 to 8, wherein the disease is Benign Prostate Hyperblasia (BPH) and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 7.
16. The method or use according to any of the claims 1 to 8, wherein the disease is Wilm's Tumor and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 9.
17. The method or use according to any of the claims 1 to 8, wherein the disease is Prostate Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 10.
18. The method or use according to any of the claims 1 to 8, wherein the disease is Lung Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 11.
19. The method or use according to any of the claims 1 to 8, wherein the disease is Pancreatic Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 12.
20. The method or use according to any of the claims 1 to 8, wherein the disease is Periodontitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 13.
21. The method or use according to any of the claims 1 to 8, wherein the disease is COPD and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 14.
22. The method or use according to any of the claims 1 to 8, wherein the disease is Ovarian Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 16.
23. The method or use according to any of the claims 1 to 8 , wherein the disease is Renal Cancer and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 3.
24. The method or use according to any of the claims 1 to 8, wherein the disease is Melanoma and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 15.
25. The method or use according to any of the claims 1 to 8, wherein the disease is Pancreatitis and said at least one platelet-miRNA comprised in the set is selected from the miRNAs listed in Figure 8.
26. The method according to any of the claims 1 to 8 or 11 to 25, wherein the determining of an expression profile of a set comprising at least one platelet-miRNA comprises the steps:
(a) reverse-transcribing the at least one platelet-miRNA comprised in the total RNA isolated into non-naturally occurring cDNA
(b) optionally amplifying the cDNA of step (a)
(c) quantifying the optionally amplified cDNA, thereby determining the expression profile of said at least one platelet-miRNA
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018151601A1 (en) * | 2017-02-17 | 2018-08-23 | Stichting Vumc | Swarm intelligence-enhanced diagnosis and therapy selection for cancer using tumor- educated platelets |
WO2019117269A1 (en) * | 2017-12-13 | 2019-06-20 | 国立大学法人広島大学 | Method for aiding detection of pancreatic cancer |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012089772A2 (en) * | 2010-12-30 | 2012-07-05 | Febit Holding Gmbh | Complex set of mirnas as non-invasive biomarkers for prostate diseases |
WO2013020926A1 (en) * | 2011-08-11 | 2013-02-14 | Febit Holding Gmbh | Complex sets of mirnas as non-invasive biomarkers for psoriasis |
WO2013026685A1 (en) * | 2011-08-19 | 2013-02-28 | Febit Holding Gmbh | Complex sets of mirnas as non-invasive biomarkers for colon cancer |
EP2578696A1 (en) * | 2011-10-06 | 2013-04-10 | febit holding GmbH | MiRNAs as non-invasive biomarkers for diagnosis |
WO2013068730A1 (en) * | 2011-11-11 | 2013-05-16 | King's College London | Micro -rnas as marker for platelet activity |
-
2014
- 2014-12-19 WO PCT/EP2014/078633 patent/WO2015091897A1/en active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012089772A2 (en) * | 2010-12-30 | 2012-07-05 | Febit Holding Gmbh | Complex set of mirnas as non-invasive biomarkers for prostate diseases |
WO2013020926A1 (en) * | 2011-08-11 | 2013-02-14 | Febit Holding Gmbh | Complex sets of mirnas as non-invasive biomarkers for psoriasis |
WO2013026685A1 (en) * | 2011-08-19 | 2013-02-28 | Febit Holding Gmbh | Complex sets of mirnas as non-invasive biomarkers for colon cancer |
EP2578696A1 (en) * | 2011-10-06 | 2013-04-10 | febit holding GmbH | MiRNAs as non-invasive biomarkers for diagnosis |
WO2013068730A1 (en) * | 2011-11-11 | 2013-05-16 | King's College London | Micro -rnas as marker for platelet activity |
Non-Patent Citations (6)
Title |
---|
ANDREAS KELLER ET AL: "Toward the blood-borne miRNome of human diseases", NATURE METHODS, NATURE PUBLISHING GROUP, GB, vol. 8, no. 10, 1 September 2011 (2011-09-01), pages 841 - 843, XP002696517, ISSN: 1548-7105, DOI: 10.1038/NMETH.1682 * |
B. VOGEL ET AL: "Multivariate miRNA signatures as biomarkers for non-ischaemic systolic heart failure", EUROPEAN HEART JOURNAL, vol. 34, no. 36, 2 September 2013 (2013-09-02), pages 2812 - 2823, XP055145967, ISSN: 0195-668X, DOI: 10.1093/eurheartj/eht256 * |
JANA SCHMITT ET AL: "Treatment-independent miRNA signature in blood of wilms tumor patients", BMC GENOMICS, BIOMED CENTRAL LTD, LONDON, UK, vol. 13, no. 1, 7 August 2012 (2012-08-07), pages 379, XP021138557, ISSN: 1471-2164, DOI: 10.1186/1471-2164-13-379 * |
KELLER ANDREAS ET AL: "miRNAs in lung cancer - Studying complex fingerprints in patient's blood cells by microarray experiments", BMC CANCER, BIOMED CENTRAL, LONDON, GB, vol. 9, no. 1, 6 October 2009 (2009-10-06), pages 353, XP021062692, ISSN: 1471-2407, DOI: 10.1186/1471-2407-9-353 * |
KELLER ANDREAS ET AL: "Multiple Sclerosis: MicroRNA Expression Profiles Accurately Differentiate Patients with Relapsing-Remitting Disease from Healthy Controls", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, US, vol. 4, no. 10, 13 October 2009 (2009-10-13), pages e7440 - 1, XP002596072, ISSN: 1932-6203, [retrieved on 20091013], DOI: 10.1371/JOURNAL.PONE.0007440 * |
VOELLENKLE CHRISTINE ET AL: "MicroRNA signatures in peripheral blood mononuclear cells of chronic heart failure patients", PHYSIOLOGICAL GENOMICS, AMERICAN PHYSIOLOGICAL SOCIETY, US, vol. 42, no. 3, 1 August 2010 (2010-08-01), pages 420 - 426, XP008133127, ISSN: 1094-8341, [retrieved on 20100518], DOI: 10.1152/PHYSIOLGENOMICS.00211.2009 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018151601A1 (en) * | 2017-02-17 | 2018-08-23 | Stichting Vumc | Swarm intelligence-enhanced diagnosis and therapy selection for cancer using tumor- educated platelets |
WO2019117269A1 (en) * | 2017-12-13 | 2019-06-20 | 国立大学法人広島大学 | Method for aiding detection of pancreatic cancer |
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