KR101758428B1 - METHOD FOR DETECTING EXOSOME miRNA USING MOLECULAR BEACON - Google Patents

Abstract

The present invention relates to a method for detecting a gene present in an exosome using a molecular beacon, comprising: a first step of isolating an exosome from a cell suspected of a disease of interest; In a non-disrupted exosome itself, a molecular beacon coupled with a detection label and a quencher that binds a detection label and a detection label to a base sequence that is capable of complementarily binding to a specific gene associated with the disease of interest. Into the exosome by contact with the exosome; And a third step of measuring a signal appearing in a detection label coupled to a molecular beacon when the molecular beacon is specifically bound to a gene associated with a disease of interest contained in the exosome.
The method of detecting a gene existing in exosome using the molecular beacon of the present invention can increase the speed, convenience and accuracy compared with the conventional detection method, By presenting a new diagnostic possibility for this difficult disease, it can be used as a new health diagnosis sensor for cancer, immune diseases, neurological diseases.

Description

METHOD FOR DETECTION EXOSOME miRNA USING MOLECULAR BEACON [0002]

The present invention relates to a method for detecting a target gene using a molecular beacon, and more particularly, to a method for detecting a target miRNA using a molecular beacon in a state in which the exosome is not disrupted.

Intercellular communication occurs through cell contact and secretion of specific molecules. The extracellular endoplasmic reticulum is divided into Exosome and Micorovesicle. Exosome is a part of the endoplasmic reticulum in the multi-vesicular body which occurs in endosome form. . Cell-derived exosomes include various proteins, fats, mRNAs and miRNAs present in cells. Exosomes are known to play an important role in signal transduction to external cells and have endocrine effects, such as migration through blood vessels and delivery to other cells at distant sites. In addition, exosomes are closely related physiologically or immunologically, such as cancer metastasis, immunity, tissue regeneration, and neurological diseases. Therefore, there is a great interest in the development of technology for treating diseases and diagnosis of diseases using exosomes, and it is expected that there will be a need for development of diagnostic methods using exosomes for diseases in which early diagnosis is difficult .

However, the progress of the study is still low compared with the importance of exosomes. However, it is difficult to isolate and purify exosomes, and the conventional method of detecting genes in exosomes is quite time- and labor- And cost-intensive processes.

Korean Patent Publication No. 10-2010-0075524

In order to analyze the miRNA contained in the purified exosome, exosome is pulverized to isolate RNA, and then cDNA is synthesized from RNA. Then, And it is necessary to quantitatively quantitate it through real-time PCR. Therefore, there is a problem that it takes much time and cost.

Accordingly, the present inventors have sought to derive a novel method for efficiently and rapidly detecting miosis-related miRNAs present in exosomes, and as a result, they have found that they are present in exosomes based on molecular beacons, which are nano-sized oligonucleotide probes (MiRNA) detection and quantification technology, which was implemented by incorporating Live Cell miRNA Imaging technology.

In short, the technical problem to be solved by the present invention is to provide a detection method for detecting miRNA existing in exosome in a live exosome itself which is not destroyed based on molecular beacon.

The problems of the present invention are not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the above object, the present invention provides a method for isolating exosomes from a cell suspected of a disease of interest, In a non-disrupted exosome itself, a molecular beacon coupled with a detection label and a quencher that binds a detection label and a detection label to a base sequence that is capable of complementarily binding to a specific gene associated with the disease of interest. Into the exosome by contact with the exosome; And a third step of measuring a signal appearing on a detection label bound to a molecular beacon when the molecular beacon is specifically bound to a gene associated with a disease of interest contained in the exosome. molecular beacon) to detect the gene present in the exosome.

The method of detecting a gene of interest using the molecular beacon of the present invention can increase the speed, convenience, and accuracy as compared with the conventional detection method.

In addition, it can be used as a new health diagnosis sensor for cancer, immune diseases, neurological diseases, etc., by presenting a new diagnosis possibility for a disease which is difficult to diagnose early through a diagnosis method of various diseases.

The effects according to the present invention are not limited by the contents exemplified above, and more various effects are included in the specification.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of fluorescence emission by complementary binding of a molecular beacon (MB) and a target miRNA according to an embodiment of the present invention. FIG.
FIG. 2 is a graph according to an embodiment of the present invention. FIG. 2 is a graph showing the relationship between the number of miRNAs detected by the miR-21 molecular beacon after the isolation of the exosome of MCF-7 cells using a total isolation kit, It is a bar graph after time lapse.
FIG. 3 is a graph according to an embodiment of the present invention. FIG. 3 is a graph showing the results of the detection of miRNA using miR-21 molecular beacon after isolation of exosomes of MCF-7 cells using a total isolation kit. It is a bar graph after time lapse.
FIG. 4 is a graph according to a control example in comparison with an embodiment of the present invention. The exosomes of CHO-K1 cells were isolated using a total exosome isolation kit and miRNA was detected using miR-21 MB ≪ / RTI > after 2 hours of reaction time.
FIG. 5 shows the results of detection using the miR-21 MB of exosome miRNA-21 isolated using ultrahigh-speed centrifugation according to an embodiment of the present invention. Panel (a) It is a bar graph after 2 hours of reaction.
6 shows the results of detection of miRNA-21 (reaction time 1 hour) using miR-21 MB after isolating exosomes of MCF-7 cells using a total exosome isolation kit according to an embodiment of the present invention, The gray bar shows the signal strength when the beacon is carried without the SLO, and the black bar is the bar graph showing the signal strength when the SLO is used for transport.
7 shows the results of detection of miRNA-21 (reaction time 2 hours) using miR-21 MB after isolating exosomes of MCF-7 cells using a total exosome isolation kit according to an embodiment of the present invention, The gray bar shows the signal strength when the beacon is carried without the SLO, and the black bar is the bar graph showing the signal strength when the SLO is used for transport.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention and the manner of achieving them will become apparent with reference to the embodiments described in detail below with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

According to one aspect of the present invention, there is provided a method for isolating exosomes from a cell suspected of having a disease of interest, In a non-disrupted exosome itself, a molecular beacon coupled with a detection label and a quencher that binds a detection label and a detection label to a base sequence that is capable of complementarily binding to a specific gene associated with the disease of interest. Into the exosome by contact with the exosome; And a third step of measuring a signal appearing on a detection label bound to a molecular beacon when the molecular beacon is specifically bound to a gene associated with a disease of interest contained in the exosome. a method for detecting a gene existing in exosome using molecular beacon is provided.

Cells suspected of having a disease of interest of the present invention may be all cells associated with exosomal secretion. Specific examples thereof include, but are not limited to, those selected from the group consisting of MCF-7, COS-7, 297-T, HeLa and NIH3T3.

In addition, subjects diagnosed using exosomes can be any type of cancer that is diagnosed, monitored, or profiled. Specific examples thereof include lung cancer, ovarian cancer, cervical cancer, endometrial cancer, breast cancer, brain cancer, colon cancer, prostate cancer, gastrointestinal cancer, head and neck cancer, non- small cell lung cancer, neural cancer, renal cancer, Pancreatic cancer, reproductive-urinary tract cancer, gallbladder cancer, melanoma, or leukemia. It can also be equally applied after detection, diagnosis and example of non-malignant tumors such as neurofibromatosis, meningiomas or schwannomas. In one embodiment of the present invention, exosomes for the diagnosis of breast cancer are targeted for diagnosis. In addition, it can equally be applied after the detection, diagnosis and example of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

As used herein, the term " target gene " means the sequence to be finally detected as " target nucleic acid ", " target nucleic acid sequence " or " target sequence ". The nucleic acid sequence includes, but is not limited to, DNA, mRNA, miRNA, snoRNA, snRNA, rRNAs, tRNAs, siRNA, hnRNA, shRNA. In some embodiments, the nucleic acid comprises one or more miRNAs. Here, the miRNA may be, for example, miR-21, miR-205, miR-92, miR-147, miR-141 or miR-574. The term " complementary " means that under certain annealing conditions or stringent conditions the primer or probe is sufficiently complementary to selectively hybridize to the target nucleic acid sequence, and the terms " substantially complementary & Perfectly complementary " as used herein, and preferably means completely complementary.

The detection label may be selected from the group consisting of enzymes, minerals and ligands, but is not necessarily limited thereto. When an enzyme is used as the detection label, available enzymes include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine Glucoamylase, terazo, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructoketase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decar A β-lactamase, and the like, but the present invention is not limited thereto.

The minerals include, but are not necessarily limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluorescamine and the like. The ligand includes, but is not necessarily limited to, biotin derivatives. Luminescent substances include, but are not necessarily limited to, acridinium ester, luciferin, luciferase, and the like.

A detection label according to an embodiment of the present invention is a detection of a fluorescent substance and emits fluorescence when a fluorescent substance bound to a molecular beacon is complementarily bound to a miRNA of interest contained in the exosome. Various methods can be used for the detection of the fluorescent material depending on the type of the fluorescent material. For example, when the fluorescent material is a fluorescent protein, the fluorescence intensity generated by irradiating ultraviolet light can be measured using a fluorophotometer. Alternatively, when the light-generating protein is luciferase, light is generated using luciferin and ATP, and the luminosity can be measured using a luminometer.

As used herein, a "molecular beacon" is an oligonucleotide that forms a hairpin-type secondary structure tagged with a quencher at the 3 'end, and the molecular beacon probe is an annealing process Hybridizes specifically in the region complementary to the template DNA, and the distance between the fluorescent substance and the fluorescent substance becomes far away, and the suppression of the emission of light by the fluorescent substance is released and fluorescence is emitted. On the other hand, since the non-hybridized molecular beacon maintains the secondary structure, it is suppressed by fluorescence and is not fluorescent.

In one embodiment of the present invention, the fluorescent substance is bound to the 5'-end of a specific base sequence, and the 3'-end is bound to the 5'-end. For example, 3'-DABCYL (4- (4-dimethyl-aminophenylazo) benzoic acid or Black Hole Quencher) can be selected as appropriate for binding to a specific fluorescent material. Fluorescein, Cy3, Cy5, Texas red, and the like can be used as the fluorescent substance reactors.

Hereinafter, the present invention will be described more specifically by way of examples. However, the following examples are provided to aid understanding of the present invention, and the scope of the present invention is not limited to these examples in any sense.

Example  One. Exosomatic  Cell culture for isolation

1) Human breast cancer cell line MCF-7 (Michigan Cancer Foundation-7) cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% FBS (Fetal Bovine Serum). It is known that microRNA-21 (miRNA-21) is highly expressed in MCF-7.

2) When the cells grew by 70% to 80%, the existing medium was removed, washed three times with 1X PBS, and then replaced with DMEM medium containing FBS with exosome removed. This is because there are a myriad of exosomes in the FBS, so that only the exosome from the MCF-7 cells can be isolated.

3) Production method of FBS without exosome

First, FBS was transferred to a polyallomer tube capable of ultra-high-speed centrifugation. This was centrifuged at 120,000 g at 4 ° C for 10 hours using a high-speed centrifuge (Beckman coulter, Optima ^™ , TLA-100.3). The supernatant was filtered using a 0.2 탆 syringe filter. The filtered exosome-free FBS was stored at 4 ° C and added to the medium at 10% as needed.

4) After replacing the cells with DMEM medium containing FBS with exosome removed, the cells were further incubated at 37 ° C for 48 hours in a CO ₂ incubator, and then the medium was collected and used for exosome isolation.

5) In order to isolate exosome from CHO-K1 (Chinese Hamster Ovary-K1) cells as a control without miRNA-21 for MCF-7 cells, experiments were also conducted. Culture of CHO-K1 cells was performed as follows. CHO-K1 cells were cultured in IMDM (Iscove's modified dulbecco's medium) medium. When the cells grew 70% to 80%, the old medium was removed, washed three times with PBS, and then replaced with IMDM medium containing 10% FBS without exosome.

6) After replacing the medium, the cells were further cultured in a CO ₂ incubator at 37 ° C for 48 hours, and then the medium was collected and used for exosome isolation.

Example  2. From the cell culture medium Exosomatic  detach

1) Exosomes were isolated from MCF-7 or CHO-K1 cells using a high-speed centrifuge or a total exosome isolation kit from Invitrogen (from cell culture media). Each method of isolating exosome is as follows.

2) Total exosome isolation kit

The medium collected from MCF-7 or CHO-K1 cell cultures cultured in T75 culture flask was centrifuged at 2,000 g at 4 ° C for 30 minutes. The supernatant was mixed with total exosome isolation reagent (Invitrogen, Cat. No. 4478359) at a ratio of 2: 1. After mixing well to make a homogeneous solution, it was incubated with O / N at 4 ° C. The next day, centrifugation was carried out at 4 ° C and 10,000 g for 1 hour. The supernatant was completely removed as completely as possible. 100 [mu] l of PBS was added to the pellet with exosomes and resuspended.

3) How to use ultra-high speed centrifuge

The MCF-7 cell culture medium was transferred to a 50 ml centrifuge tube and centrifuged at 300 g for 10 minutes at 4 ° C. The supernatant was transferred to a new tube and centrifuged again at 2,000 xg for 10 minutes. The supernatant was transferred to a polyallomer tube capable of ultra-high speed centrifugation. This was centrifuged at 100,000 g at 4 ° C for 2 hours using a high-speed centrifuge (Beckman coulter, Optima ^™ , TLA-100.3) and the supernatant was removed as completely as possible. To wash the pellet with exosomes, 3 ml PBS was added to the tube and resuspended. And then centrifuged at 100,000 g for 2 hours at 4 ° C. The supernatant was removed as completely as possible. The pellet with exosomes was resuspended by adding 100 [mu] l of PBS and stored at 4 [deg.] C or -20 [deg.] C.

Example  3. Molecules The beacon (molecular beacon)  Used Exosome  of mine miRNA -21 detection

1) A molecular beacon was used to detect the microRNA (miRNA) inside the exosome in a normal intact exosome state without breaking the exosome. A molecular beacon has a fluorescent substance and a quencher at both ends. Cy3, a positive charge, was used as a fluorescent material, and BHQ2 was used as a Cy3 quencher.

2) Molecular beacon (miR-21 MB) specifically binding to miRNA-21 was synthesized (Integrated DNA Technologies, Inc) to directly detect miRNA-21 in exoose isolated from MCF-7 cell culture Respectively. The sequence of MiR-21 MB is shown in Table 1 below, and the manner in which the molecular beacon (MB) binds to the miRNA is shown in FIG. When a binding reaction is carried out with a molecular beacon (MB) having a sequence complementary to a target miRNA, the fluorescent signal of the MB fluorescer increases as the distance from the quencher increases.

Molecular beacon Molecular beacon sequence SEQ ID NO: 1
miR-21 MB Cy3-GCGCGTCAACATCAGTCTGATAAGCTACGCGC-BHQ2

3) First, each of the exosomes isolated by the above-described exosome isolation method (total exosome isolation kit and ultra-high speed centrifugation) was diluted to final concentrations 1/10, 1/20, 1/40, 1/80 (Total exosome isolation kit) or 1/5, 1/10 (ultra-high-speed centrifugation method). The different dilution rates of exosomes used in each method are different because the amount of exosome isolated by each method is different.

4) MiR-21 MB was adjusted to a final concentration of 100 nM. When dealing with the MiR-21 MB, the light was blocked as much as possible.

5) As a control group for obtaining a background signal (noise), which is a nonspecific fluorescence signal, a solution in which only exocose was added but only molecular beacon was added was used (1 in Table 2). In the comparative groups (2 to 5), different concentrations of exosomes diluted sequentially were added and 1/10 of the diluted solutions (A, B, C, and D) ), 1/20 (3 in Table 2), 1/40 (4 in Table 2), and 1/80 (5 in Table 2) were used.

45 μl of each mixed solution was transferred to a 384-well black microplate. After blocking the light, the reaction was continued for 2 hours at 37 ° C. The reaction was carried out at 1 hour intervals using a fluorescence measuring device (Thermo scientific, Varioskan ™, Flash Multimode Reader) Fluorescence was measured. The composition of the solution for detecting exosome miRNA-21 is shown in Table 2 below.

One 2 3 4 5 MB MB
E1 / 10 MB
E1 / 20 MB
E1 / 40 MB
E1 / 80 MB (1 μM) (μl) 5 5 5 5 5 Exosome (μl) 0 5 from A 5 from B 5 from C 5 from D PBS (ll) 45 40 40 40 40 Total (μl) 50 50 50 50 50

A: Exosome raw solution

B: The stock solution was diluted 1: 1 with 1X RNase free PBS

C: B diluted 1: 1 with 1X RNase free PBS

Dilute D: C with 1X RNase free PBS at a ratio of 1: 1

3-1. Experimental Results Experimental Results - MCF-7 Cell Experiment

1) The fluorescence signal measured in each sample was expressed as a signal to background value divided by the fluorescence value (background) of the first sample (control) without the exosome.

2) Figures 2 and 3 show the results of detection of miRNA-21 in exosome isolated from MCF-7 cells using miR-21 MB using Invitrogen's total exosome isolation kit.

As shown in FIG. 2, which is the result after one hour of the reaction, it can be seen that the fluorescence signal increases with miR-21 MB as the concentration of exosomes increases. This suggests that the higher the concentration of MCF-7 exosomes, the greater the concentration of miRNA-21 in exosomes and the greater the amount of miR-21 MB associated with them. In FIG. 3, as the reaction time becomes 2 hours, the time for miR-21 MB to enter the exosome and the time for binding to miRNA-21 in the exosomes are increased, and the signal is significantly increased from 1 hour. In addition, we can observe that the signal to background value also increases as the concentration of exosomes increases.

3-2. Control experiments - Experiments in CHO-K1 cells

1) In order to confirm that the signals from FIGS. 2 and 3 specifically bind miRNA-21 and miR-21 MB in the exosome, the same method was used to extract exosomes from CHO-K1 cells without miRNA- After the separation, the same experiment was carried out. miRNA-21 is a miRNA that exists only in human cells, and CHO-K1 does not have miRNA-21 as a hamster-derived cell.

4, the fluorescence signal of the CHO-K1 exosome without miRNA-21 is significantly weaker than that of the signal of FIG. 3 even after 2 hours of the reaction. This shows that the amplification of the fluorescence signal shown in FIGS. -21 MB and exosome miRNA-21. Therefore, it is possible to distinguish the presence or absence of miRNA-21 in the exosome and the concentration using molecular beacons.

3-3. As a result of the control experiment, 2-speed ultracentrifugation

Exosomes were isolated from MCF-7 cells by ultrafast centrifugation, and then exosome miRNA-21 was detected using miR-21 MB (FIG. 5). As a result of analyzing the detection signals of miR-21 MB by diluting the concentrations of the separated exosomes to 1/5 and 1/10, respectively, it was confirmed that miR-21 MB was obtained by using the total exosome isolation kit of Invitrogen It can be seen that the fluorescence signal increases as the concentration of exosomes increases. Also, it can be seen that the fluorescence signal increases as the reaction time becomes longer.

Therefore, it can be confirmed that the method of detecting miRNA in exosome from a cell using a molecular beacon can specifically detect miRNA regardless of the method of isolating exosome. In addition, it can be confirmed that the fluorescence signal increases as the amount of exosome, that is, the amount of miRNA which is a binding target of molecular beacon increases.

This is significantly simpler and more convenient than the method of isolating miRNA from exosomes and synthesizing cDNA from isolated miRNA and amplifying it by real-time PCR, and is also excellent in terms of cost and time (for example, , 13 hours → 1 hour).

4-1. Activation of SLO (Streptolysin O) for the delivery of MiR-21 MB in exosomes

1) An attempt was made to carry more molecular beacons into the exosomes to further improve the detection of miRNAs in exosomes using the molecular beacons described above. Comparing the results in FIGS. 2 and 3, it can be seen that as the reaction time increases, the combined fluorescence signal of the molecular beacon and miRNA increases. This is probably because the amount of beacon that can bind to miRNA in exosomes increases as the amount of molecular beacon carried into exosomes increases. Therefore, it is possible to detect the exosomal miRNA more rapidly by carrying the molecular beacon to the exosomes more rapidly, and therefore, the present invention uses Streptolysin O (SLO) for this purpose.

2) Plasma membrane of animal cells contains a lot of cholesterol. Exosomes also have membranes similar to cell membrane components, and there is also a large amount of cholesterol. SLO sticks to the cholesterol and pushes the cholesterol to pierce the cell surface. Thus, using SLOs to drill holes in exosomes, it would be possible to increase miRNA detection efficiency if molecular beacons can be transported smoothly into the exosome.

3) On the other hand, since SLO is easily oxidized to lose its activity, TCEP (Tris (2-carboxyethyl) phosphine hydrochloride), a reducing reagent, was used to maintain the activity of SLO.

4) 15 mg of TCEP was dissolved in 50 μl of water and vigorously stirred.

5) 200 μl of 2 U / ml SLO and 1 μl of TCEP prepared above were mixed and the activation reaction was carried out at 37 ° C for 1 hour. The SLO solution thus prepared was used in the following exosomal experiments.

4-2. Experimental results - Confirmation of improvement of detection efficiency of miRNA-21 in exosome of molecular beacon using SLO

1) Exosomes isolated by Invitrogen's total exosome isolation kit described in Example 2 were diluted with PBS to final concentrations 1/10, 1/20, 1/40, and 1/80. MiR-21 MB was used at 1 μM to achieve a final concentration of 100 nM and blocked as much as possible during the experiment.

2) SLO was activated by using TCEP and the final concentration was adjusted to 0.2 U / mL.

3) Table 3 shows the composition of the solution for detecting exosome miRNA-21. As a control group for obtaining a background signal (noise), which is a nonspecific fluorescence signal, a solution containing only exogenous molecular beacon was used 3 of 1). In the comparative groups (2 to 5, 7 to 10), sequentially diluted different concentrations of exosomes were added, and 1/10 of the exosomes (A, B, C and D) 2 and 7 in Table 3), 1/20 (3 and 8 in Tables 3), 1/40 (4 and 9 in Tables 3), 1/80 (5 and 10 in Tables 3) were used.

One 2 3 4 5 6 7 8 9 10 MB MB
E1 / 10 MB
E1 / 20 MB
E1 / 40 MB
E1 / 80 MB
SLO MB
SLO
E1 / 10 MB
SLO
E1 / 20 MB
SLO
E1 / 40 MB
SLO
E1 / 80 MB (100 nM) (占 퐇) 5 5 5 5 5 5 5 5 5 5 Exosome (μl) 0 5 from A 5 from B 5 from C 5 from D 0 5 from A 5 from B 5 from C 5 from D SLO (0.2 U / mL)
(Μl) 0 0 0 0 0 5 5 5 5 5 RNase-free PBS (占 퐇) 45 40 40 40 40 40 35 35 35 35 Total
(Μl) 50 50 50 50 50 50 50 50 50 50

A: Exosome raw solution

B: The stock solution was diluted 1: 1 with 1X RNase free PBS

C: B diluted 1: 1 with 1X RNase free PBS

Dilute D: C with 1X RNase free PBS at a ratio of 1: 1

45 μl of each mixed solution was transferred to a 384-well black microplate. After blocking the light, the reaction was continued for 2 hours at 37 ° C. Fluorescence was measured at intervals of 1 hour using a fluorescence measuring device (Thermo scientific, Varioskan ™, Flash Multimode Reader).

4) The values 1 to 5 in Table 3 are the same as those described in Figs. 2 and 3, and the fluorescence value measured in each of the 6th to 10th samples was compared with the first sample (control) without the exosome, And the signal-to-background value divided by the fluorescence value (background). FIG. 6 shows the results of detection of miRNA-21 (reaction time 1 hour) using miR-21 MB after isolation of exosome of MCF-7 cells using a total exosome isolation kit. FIG. 7 shows the result when the reaction time of 2 hours has elapsed in FIG. 6. FIG.

5) As shown in FIG. 6, which is the result after 1 hour of the reaction, the signal to background value was significantly increased when beacons were punched through the holes in the exosomes by adding SLO to the beacons carried in the exosomes without SLO have. In one example, when using 1/10 diluted exosomes (2 and 7 in Table 3), the signal to background value was 6.485 without SLO, whereas the value increased to more than double when added with SLO can see.

6) This indicates that the pathway through which the beacon is transported by the SLO occurs in the exosome membrane, which increases the amount of beacon transported into the exosome, thereby increasing the amount and speed of binding with miRNA-21.

7) As shown in FIG. 7, after 2 hours of the reaction, the signal to background value was greatly increased by SLO as in FIG. 6. In addition, the intensity of the fluorescence signal is increased compared to the reaction time of 1 hour. When 1/10 diluted exosomes were used, the signal to background value was 16.07 without SLO, whereas the SLO increased to more than doubled to 29.65. This value is 2.2 times higher than 13.47 of 1 hour.

8) This is because SLO causes more holes in the exosomal membrane as time passes, and also increases the amount of beacons to be transported and increases the amount of beacon bound to miRNA-21.

9) In conclusion, SLO can effectively increase beacon delivery to exosomes, which can shorten the detection time of miRNA in exosomes and increase the sensitivity of the assay.

10) Therefore, the present invention shows that a specific miRNA in the exosome can be efficiently detected using a molecular beacon as a technique for diagnosing disease through detection of a specific miRNA in the exosome, without exocomosing the exosome, SLO can improve the detection efficiency and sensitivity of exosome miRNA effectively.

Claims (8)

A method for detecting a gene in an exosome from a cell,
A first step of isolating exosomes from cells suspected of the disease of interest;
In a non-disrupted exosome itself, a molecular beacon (quencher) coupled with a detection label and a detection label at the end of the nucleotide sequence capable of complementarily binding with miRNA, a specific gene associated with the disease of interest molecular beacon) into the exosome by contacting the exosome with the exosome; And
And a third step of measuring a fluorescence signal appearing on a detection label bound to a molecular beacon when the miRNA contained in the exosome and the molecular beacon are specifically bound to each other,
The first step includes culturing cells suspected of the disease of interest in a medium containing FBS (Fetal Bovine Serum), wherein the cells are cultured in an initial medium, Lt; RTI ID = 0.0 > FBS, < / RTI >
In the second step, the contact between the molecular beacon and the exosome is carried out by adding and mixing exosomes to a solution containing a molecular beacon, wherein SLO (Streptolysin O) and TCEP (Tris (2-carboxyethyl) phosphine hydrochloride) The molecular beacon is contacted with the exosomes under mixed mixed solution,
The SLO is activated by the TCEP to push out cholesterol present in the exosome membrane and to pierce the exosome membrane to increase the efficiency of transport of the molecular beacon into the exosome.
The suspected disease-suspected cell is a human breast cancer cell line MCF-7 (Michigan Cancer Foundation-7)
The miRNA is miRNA-21,
The molecular beacon includes a positively charged Cyanine series fluorescent substance and has a nucleotide sequence shown in SEQ ID NO: 1 below,
In the first step, the isolation of exosomes is performed using a total exosome isolation kit,
In the second step, the solution used for contacting the molecular beacon with the exosome is a solution containing exosome diluted with PBS (Phosphate Buffer Saline), wherein the concentration of the molecular beacon is 100 nM, the concentration of exosome is 10 vol%, a concentration of SLO of 0.2 U / mL,
In the second step, the contact reaction between the molecular beacon and the exosome is carried out for 2 hours with the light blocked.
Methods for detecting genes present in exosomes using molecular beacons:
[SEQ ID NO: 1]
Cy3-GCGCGTCAACATCAGTCTGATAAGCTACGCGC-BHQ2. delete delete delete delete delete delete delete

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