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WO2006048273A1 - Procedes de validation des tests d'expression genique - Google Patents

Procedes de validation des tests d'expression genique Download PDF

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Publication number
WO2006048273A1
WO2006048273A1 PCT/EP2005/011749 EP2005011749W WO2006048273A1 WO 2006048273 A1 WO2006048273 A1 WO 2006048273A1 EP 2005011749 W EP2005011749 W EP 2005011749W WO 2006048273 A1 WO2006048273 A1 WO 2006048273A1
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Prior art keywords
gene expression
cells
expression
rna
genes
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PCT/EP2005/011749
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English (en)
Inventor
Torsten Haferlach
Martin Dugas
Wolfgang Kern
Alexander Kohlmann
Susanne Schnittger
Claudia Schoch
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Roche Diagnostics Gmbh
F.Hoffmann-La Roche Ag
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Publication of WO2006048273A1 publication Critical patent/WO2006048273A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia

Definitions

  • the present invention relates to gene expression analyses and to methods of validating such analyses.
  • Leukemias are generally classified into four different groups or types: acute myeloid (AML), acute lymphatic (ALL), chronic myeloid (CML) and chronic lymphatic leukemia (CLL). Within these groups, several subcategories or subtypes can be identified using various approaches. These different subcategories of leukemia are associated with varying clinical outcomes and therefore can serve as guides to the selection of different treatment strategies. The importance of highly specific classification may be illustrated for AML as a very heterogeneous group of diseases. Effort has been aimed at identifying biological entities and to distinguish and classify subgroups of AML that are associated with, e.g., favorable, intermediate or unfavorable prognoses.
  • the FAB classification was proposed by the French- American-British co-operative group that utilizes cytomorphology and cytochemistry to separate AML subgroups according to the morphological appearance of blasts in the blood and bone marrow.
  • genetic abnormalities occurring in leukemic blasts were recognized as having a major impact on the morphological picture and on prognosis.
  • the karyotype of leukemic blasts is commonly used as an independent prognostic factor regarding response to therapy as well as survival.
  • a combination of methods is typically used to obtain the diagnostic information in leukemia.
  • the analysis of the morphology and cytochemistry of bone marrow blasts and peripheral blood cells is commonly used to establish a diagnosis.
  • immunophenotyping is also utilized to separate an undifferentiated AML from acute lymphoblastic leukemia and from CLL.
  • leukemia subtypes can be diagnosed by cytomorphology alone, but this typically requires that an expert review sample smears.
  • genetic analysis based on, e.g., chromosome analysis, fluorescence in situ hybridization (FISH), or reverse transcription PCR (RT-PCR) and immunophenotyping is also generally used to accurately assign cases to the correct category.
  • FISH fluorescence in situ hybridization
  • RT-PCR reverse transcription PCR
  • An aim of these techniques, aside from diagnosis, is to determine the prognosis of the leukemia under consideration.
  • One disadvantage of these methods is that viable cells are generally necessary, as the cells used for genetic analysis need to divide in vitro in order to obtain metaphases for the analysis.
  • Another exemplary problem is the long lag period (e.g., 72 hours) that typically occurs between the receipt of the materials to be analyzed in the laboratory and the generation of results.
  • great experience in preparing chromosomes and analyzing karyotypes is generally needed to obtain correct results in most cases.
  • hematological malignancies can be separated into CML, CLL, ALL, and AML. Within the latter three disease entities, several prognostically relevant subtypes have been identified. This further sub-classification commonly relies on genetic abnormalities of leukemic blasts and is associated with different prognoses.
  • the sub-classification of leukemias is used increasingly as a guide to the selection of appropriate therapies.
  • the development of new, specific drugs and treatment approaches often includes the identification of specific subtypes that may benefit from a distinct therapeutic protocol and thus, improve the outcomes of distinct subsets of leukemia.
  • the therapeutic drug (STI571) inhibits the CML specific chimeric tyrosine kinase BCR-ABL generated from the genetic defect observed in CML, the BCR-ABL-rearrangement due to the translocation between chromosomes 3 and 22 (t(9;22) (q34;qll)).
  • the therapy response is dramatically higher as compared to other drugs that have previously been used.
  • AML M3 and its variant M3v are subtypes of acute myeloid leukemia, AML M3 and its variant M3v, which both include the karyotype t(15;17)(q22;qll- 12).
  • ATRA all-trans retinoic acid
  • Golub et al. Science, 1999, 286, 531-7, which is incorporated by reference
  • gene expression profiles can be used for class prediction and discriminating AML from ALL samples.
  • the present invention relates to rapid, cost effective, and reliable approaches to validating or assessing the robustness of a given gene expression analysis.
  • the invention provides methods of validating a diagnostic gene expression assay.
  • the methods include (a) providing a population of cells and (b) subjecting at least two sub-populations of the cells to at least one different assay parameter.
  • the population of cells comprises one or more of, e.g., acute myeloid leukemia (AML) cells, acute lymphatic leukemia (ALL) cells, chronic myeloid leukemia (CML) cells, or chronic lymphatic leukemia (CLL) cells.
  • AML acute myeloid leukemia
  • ALL acute lymphatic leukemia
  • CML chronic myeloid leukemia
  • CLL chronic lymphatic leukemia
  • the methods also include (c) detecting expression levels of substantially identical sets of genes in or derived from the cells in the sub- populations to produce at least two gene expression data sets and (d) determining whether the gene expression data sets are substantially similar to one another, thereby validating the diagnostic gene expression assay.
  • the methods include (e) selecting the gene expression data sets that are substantially similar to one another, and optionally (f) using the selected gene expression data sets in performing the diagnostic gene expression assay.
  • the methods include (e) repeating (b) - (d) using a modified assay parameter, if the gene expression data sets are not substantially similar to one another.
  • any assay parameter or set of parameters can be assessed according to these validation methods.
  • Exemplary assay parameters include one or more of, e.g., a duration of sample shipment time; a measure of RNA and/or protein quality; a duration of sample storage time prior to target preparation; a date of sample target preparation within a marker discovery study; an age of a subject at diagnosis; an assay operator; a washing condition; a detection condition; a specimen type; or the like.
  • the measure of RNA quality optionally comprises a 375' ratio of GAPD hybridization signals.
  • the specimen type comprises a bone marrow sample or a peripheral blood sample in certain embodiments.
  • Expression levels are detected using essentially any gene expression profiling technique.
  • the expression level is detected using an array, a robotics system, and/or a microfluidic device.
  • the expression level of the set of genes is detected by amplifying nucleic acid sequences associated with the genes to produce amplicons and detecting the amplicons.
  • the amplicons are generally detected using a process that comprises one or more of: hybridizing the amplicons to an oligonucleotide array, digesting the amplicons with a restriction enzyme, or real-time polymerase chain reaction (PCR) analysis.
  • PCR real-time polymerase chain reaction
  • the expression level of the set of genes is detected by, e.g., measuring quantities of transcribed polynucleotides (e.g., mRNAs, cDNAs, etc.) or portions thereof expressed or derived from the genes. In some embodiments, the expression level is detected by, e.g., contacting polynucleotides or polypeptides expressed from the genes with compounds (e.g., aptamers, antibodies or fragments thereof, etc.) that specifically bind the polynucleotides or polypeptides.
  • compounds e.g., aptamers, antibodies or fragments thereof, etc.
  • Figures 2A-2F Analysis of varying sample handling and operator parameters. Principal component and hierarchical cluster analyses based on Affymetrix Ul 33 chip design microarray expression data. This analysis was based on a subset of genes which were identified to be differentially expressed when analyzing samples from the subtypes AML with t(15;17), inv(16), inv(3), or CML. In the three- dimensional PCA plot data points with similar characteristics will cluster together. Here, each patient's expression pattern is represented by a single color-coded sphere. The labels and coloring of the classes were added after the analysis for means for better visualization.
  • Figure 2A Differentially expressed genes between AML with t(15;17) and inv(16) from two different operators (top 300 probe sets), operator 1.
  • FIG. 2B Differentially expressed genes between AML with t(15;17) and inv(16) from two different operators (top 300 probe sets), operator 2.
  • Figure 2C Top 300 differentially expressed probe sets when combining both operators visualized by PCA.
  • Figure 2D Hierarchical Clustering using top 300 differentially expressed probe sets when combining both operators.
  • Figure 2E Differentially expressed genes between AML with inv(3) and CML. This analysis includes freshly prepared versus frozen AML with inv(3) samples. The frozen samples had been prepared after different storage times at room temperature.
  • Figure 2F Visualization of the CML samples from the previous analysis by PCA. The samples were prepared in replicates by two different operators in different laboratories.
  • Ilq23/MLL refers to acute myeloid leukemia with the 1 Iq23 rearrangement of the human MLL gene according to the World Health Organization (WHO) classification of haematological malignancies.
  • WHO World Health Organization
  • an “antibody” refers to a polypeptide substantially encoded by at least one immunoglobulin gene or fragments of at least one immunoglobulin gene, which can participate in specific binding with a ligand.
  • the term “antibody” includes polyclonal and monoclonal antibodies and biologically active fragments thereof including among other possibilities “univalent” antibodies (Glennie et al.
  • V H and V L regions typically variable heavy and light chain regions
  • CDRs complementarity determining regions
  • scFvs chimeric and humanized antibodies. See, e.g., Harlow and Lane, Antibodies, a laboratory manual. CSH Press (1988), which is incorporated by reference.
  • methods known to a person skilled in the art, which are optionally utilized. Examples include immunoprecipitations, Western blottings, Enzyme-linked immuno sorbent assays (ELISA), radioimmunoassays (RIA), dissociation-enhanced lanthanide fluoro immuno assays (DELFIA), scintillation proximity assays (SPA).
  • an antibody is typically labeled by one or more of the labels described herein or otherwise known to persons skilled in the art.
  • an "array” or “microarray” refers to a linear or two- or three dimensional arrangement of preferably discrete nucleic acid or polypeptide probes which comprises an intentionally created collection of nucleic acid or polypeptide probes of any length spotted onto a substrate/solid support.
  • a collection of nucleic acids or polypeptide spotted onto a substrate/solid support also under the term "array”.
  • a microarray usually refers to a miniaturized array arrangement, with the probes being attached to a density of at least about 10, 20, 50, 100 nucleic acid molecules referring to different or the same genes per cm 2 .
  • an array can be referred to as "gene chip”.
  • the array itself can have different formats, e.g., libraries of soluble probes or libraries of probes tethered to resin beads, silica chips, or other solid supports.
  • “Complementary” and “complementarity”, respectively, can be described by the percentage, i.e., proportion, of nucleotides that can form base pairs between two polynucleotide strands or within a specific region or domain of the two strands.
  • complementary nucleotides are, according to the base pairing rules, adenine and thymine (or adenine and uracil), and cytosine and guanine.
  • Complementarity may be partial, in which only some of the nucleic acids' bases are matched according to the base pairing rules.
  • nucleic acid strands there may be a complete or total complementarity between the nucleic acids.
  • the degree of complementarity between nucleic acid strands has effects on the efficiency and strength of hybridization between nucleic acid strands.
  • Two nucleic acid strands are considered to be 100% complementary to each other over a defined length if in a defined region all adenines of a first strand can pair with a thymine (or an uracil) of a second strand, all guanines of a first strand can pair with a cytosine of a second strand, all thymine (or uracils) of a first strand can pair with an adenine of a second strand, and all cytosines of a first strand can pair with a guanine of a second strand, and vice versa.
  • the degree of complementarity is determined over a stretch of about 20 or 25 nucleotides, i.e., a 60% complementarity means that within a region of 20 nucleotides of two nucleic acid strands 12 nucleotides of the first strand can base pair with 12 nucleotides of the second strand according to the above base pairing rules, either as a stretch of 12 contiguous nucleotides or interspersed by non-pairing nucleotides, when the two strands are attached to each other over the region of 20 nucleotides.
  • the degree of complementarity can range from at least about 50% to full, i.e., 100% complementarity.
  • Two single nucleic acid strands are said to be "substantially complementary" when they are at least about 80% complementary, and more typically about 90% complementary or higher.
  • substantial complementarity is generally utilized.
  • Two nucleic acids “correspond” when they have substantially identical or complementary sequences, when one nucleic acid is a subsequence of the other, or when one sequence is derived naturally or artificially from the other.
  • differential gene expression refers to a gene or set of genes whose expression is activated to a higher or lower level in a subject suffering from a disease, (e.g., cancer) relative to its expression in a normal or control subject. Differential gene expression can also occur between different types or subtypes of diseased cells. The term also includes genes whose expression is activated to a higher or lower level at different stages of the same disease. It is also understood that a differentially expressed gene may be either activated or inhibited at the nucleic acid level or protein level, or may be subject to alternative splicing to result in a different polypeptide product.
  • Differential gene expression may include a comparison of expression between two or more genes or their gene products, or a comparison of the ratios of the expression between two or more genes or their gene products, or even a comparison of two differently processed products of the same gene, which differ between, e.g., normal subjects and subjects suffering from a disease, various stages of the same disease, different types or subtypes of diseased cells, etc.
  • Differential expression includes both quantitative, as well as qualitative, differences in the temporal or cellular expression pattern in a gene or its expression products among, for example, normal and diseased cells, or among cells which have undergone different disease events or disease stages.
  • "differential gene expression” is considered to be present when there is at least an about two-fold, typically at least about four-fold, more typically at least about six- fold, most typically at least about ten-fold difference between, e.g., the expression of a given gene in normal and diseased subjects, in various stages of disease development in a diseased subject, different types or subtypes of diseased cells, etc.
  • expression refers to the process by which mRNA or a polypeptide is produced based on the nucleic acid sequence of a gene, i.e., "expression” also includes the formation of mRNA in the process of transcription.
  • determining the expression level refers to the determination of the level of expression of one or more markers.
  • genotype refers to a description of the alleles of a gene or genes contained in an individual or a sample. As used herein, no distinction is made between the genotype of an individual and the genotype of a sample originating from the individual. Although, typically, a genotype is determined from samples of diploid cells, a genotype can be determined from a sample of haploid cells, such as a sperm cell.
  • gene refers to a nucleic acid sequence encoding a gene product.
  • the gene optionally comprises sequence information required for expression of the gene (e.g., promoters, enhancers, etc.).
  • gene expression data refers to one or more sets of data that contain information regarding different aspects of gene expression.
  • the data set optionally includes information regarding: the presence of target-transcripts in cell or cell- derived samples; the relative and absolute abundance levels of target transcripts; the ability of various treatments to induce expression of specific genes; and the ability of various treatments to change expression of specific genes to different levels.
  • Such conditions are, for example, hybridization in 6x SSC, pH 7.0 / 0.1 % SDS at about 45 0 C for 18-23 hours, followed by a washing step with 2x SSC/1 % SDS at 50°C.
  • the salt concentration in the washing step can, for example, be chosen between 2x SSC/0.1 % SDS at room temperature for low stringency and 0.2x SSC/0.1 % SDS at 50 0 C for high stringency.
  • the temperature of the washing step can be varied between room temperature (ca. 22 0 C), for low stringency, and 65 0 C to 70 0 C for high stringency.
  • polynucleotides that hybridize at lower stringency hybridization conditions.
  • Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of, e.g., formamide concentration (lower percentages of formamide result in lowered stringency), salt conditions, or temperature.
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g., 5x SSC).
  • Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described herein, due to problems with compatibility.
  • inv(16) refers to AML with inversion 16 according to the WHO classification of haematological malignancies.
  • label refers to a moiety attached (covalently or non-covalently), or capable of being attached, to a molecule (e.g., a polynucleotide, a polypeptide, etc.), which moiety provides or is capable of providing information about the molecule (e.g., descriptive, identifying, etc. information about the molecule) or another molecule with which the labeled molecule interacts (e.g., hybridizes, etc.).
  • a molecule e.g., a polynucleotide, a polypeptide, etc.
  • information about the molecule e.g., descriptive, identifying, etc. information about the molecule
  • another molecule with which the labeled molecule interacts e.g., hybridizes, etc.
  • Exemplary labels include fluorescent labels (including, e.g., quenchers or absorbers), non- fluorescent labels, colorimetric labels, chemiluminescent labels, bioluminescent labels, radioactive labels (such as 3 H, 35 S, 32 P, 125 1, 57 Co or 14 C), mass-modifying groups, antibodies, antigens, biotin, haptens, digoxigenin, enzymes (including, e.g., peroxidase, phosphatase, etc.), and the like.
  • fluorescent labels including, e.g., quenchers or absorbers
  • non- fluorescent labels including, e.g., colorimetric labels, chemiluminescent labels, bioluminescent labels, radioactive labels (such as 3 H, 35 S, 32 P, 125 1, 57 Co or 14 C), mass-modifying groups, antibodies, antigens, biotin, haptens, digoxigenin, enzymes (including, e.g., peroxidase, phosphatase, etc.), and
  • fluorescent labels may include dyes that are negatively charged, such as dyes of the fluorescein family, or dyes that are neutral in charge, such as dyes of the rhodamine family, or dyes that are positively charged, such as dyes of the cyanine family.
  • Dyes of the fluorescein family include, e.g., FAM, HEX, TET, JOE, NAN and ZOE.
  • Dyes of the rhodamine family include, e.g., Texas Red, ROX, Rl 10, R6G, and TAMRA.
  • FAM, HEX, TET, JOE, NAN, ZOE, ROX, Rl 10, R6G, and TAMRA are commercially available from, e.g., Perkin-Elmer, Inc. (Wellesley, MA, USA), and
  • Texas Red is commercially available from, e.g., Molecular Probes, Inc. (Eugene, OR, USA). Dyes of the cyanine family include, e.g., Cy2, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7, and are commercially available from, e.g., Amersham Biosciences Corp. (Piscataway, NJ, USA). Suitable methods include the direct labeling (incorporation) method, an amino-modified (amino-allyl) nucleotide method (available e.g. from Ambion, Inc. (Austin, TX, USA), and the primer tagging method (DNA dendrirner labeling, as kit available e.g. from Genisphere, Inc.
  • biotin or biotinylated nucleotides are used for labeling, with the latter generally being directly incorporated into, e.g., the cRNA polynucleotide by in vitro transcription.
  • the term “lower expression” refers an expression level of one or more markers from a target that is less than a corresponding expression level of the markers in a reference. In certain embodiments, "lower expression” is assigned to all by numbers and Affymetrix Id. definable polynucleotides the t-values and fold change (fc) values of which are negative.
  • the term “higher expression” refers an expression level of one or more markers from a target that is more than a corresponding expression level of the markers in a reference.
  • “higher expression” is assigned to all by numbers and Affymetrix Id. definable polynucleotides the t-values and fold change (fc) values of which are positive.
  • a “machine learning algorithm” refers to a computational-based prediction methodology, also known to persons skilled in the art as a “classifier”, employed for characterizing a gene expression profile. The signals corresponding to certain expression levels, which are obtained by, e.g., microarray-based hybridization assays, are typically subjected to the algorithm in order to classify the expression profile. Supervised learning generally involves "training" a classifier to recognize the distinctions among classes and then “testing" the accuracy of the classifier on an independent test set. For new, unknown samples the classifier can be used to predict the class in which the samples belong.
  • markers refers to a genetically controlled difference that can be used in the genetic analysis of a test or target versus a control or reference sample for the purpose of assigning the sample to a defined genotype or phenotype.
  • markers refer to genes, polynucleotides, polypeptides, or fragments or portions thereof that are differentially expressed in, e.g., different leukemia types and/or subtypes.
  • the markers can be defined by their gene symbol name, their encoded protein name, their transcript identification number (cluster identification number), the data base accession number, public accession number and/or GenBank identifier.
  • Markers can also be defined by their Affymetrix identification number, chromosomal location, UniGene accession number and cluster type, and/or LocusLink accession number.
  • the Affymetrix identification number (affy id) is accessible for anyone and the person skilled in the art by entering the "gene expression omnibus" internet page of the National Center for Biotechnology Information (NCBI) on the world wide web at ncbi.nlm.nih.gov/geo/ as of 11/4/2004.
  • NCBI National Center for Biotechnology Information
  • the affy id's of the polynucleotides used for certain embodiments of the methods described herein are derived from the so-called human genome Ul 33 chip (Affymetrix, Inc., Santa Clara, CA, USA).
  • sequence data of each identification number can be viewed on the world wide web at, e.g., ncbi.nlm.nih.gov/projects/geo/ as of 1 1/4/2004 using the accession number GPL96 for U133A annotational data and accession number GPL97 for U133B annotational data.
  • the expression level of a marker is determined by the dete ⁇ nining the expression of its corresponding polynucleotide.
  • normal karyotype refers to a state of those cells lacking any visible karyotype abnormality detectable with chromosome banding analysis.
  • nucleic acid refers to a polymer of monomers that can be corresponded to a ribose nucleic acid (RNA) or deoxyribose nucleic acid (DNA) polymer, or analog thereof. This includes polymers of nucleotides such as RNA and DNA, as well as modified forms thereof, peptide nucleic acids (PNAs), locked nucleic acids (LNATMs), and the like.
  • the nucleic acid can be a polymer that includes multiple monomer types, e.g., both RNA and DNA subunits.
  • a nucleic acid can be or include, e.g., a chromosome or chromosomal segment, a vector (e.g., an expression vector), an expression cassette, a naked DNA or RNA polymer, the product of a polymerase chain reaction (PCR) or other nucleic acid amplification reaction, an oligonucleotide, a probe, a primers, etc.
  • a nucleic acid can be e.g., single-stranded or double-stranded. Unless otherwise indicated, a particular nucleic acid sequence optionally comprises or encodes complementary sequences, in addition to any sequence explicitly indicated.
  • Oligonucleotides e.g., probes, primers, etc.
  • Oligonucleotides of a defined sequence may be produced by techniques known to those of ordinary skill in the art, such as by chemical or biochemical synthesis, and by in vitro or in vivo expression from recombinant nucleic acid molecules, e.g., bacterial or retroviral vectors.
  • Oligonucleotides which are primer and/or probe sequences, as described below, may comprise DNA, RNA or nucleic acid analogs such as uncharged nucleic acid analogs including but not limited to peptide nucleic acids (PNAs) which are disclosed in International Patent Application WO 92/20702 or morpholino analogs which are described in U.S. Pat. Nos. 5,185,444, 5,034,506, and 5,142,047 all of which are incorporated by reference. Such sequences can routinely be synthesized using a variety of techniques currently available.
  • PNAs peptide nucleic acids
  • a sequence of DNA can be synthesized using conventional nucleotide phosphoramidite chemistry and the instruments available from Applied Biosystems, Inc, (Foster City, CA, USA); DuPont, (Wilmington, DE, USA); or Milligen, (Bedford, MA, USA).
  • the sequences can be labeled using methodologies well known in the art such as described in U.S. patent application numbers 5,464,746;
  • a nucleic acid, nucleotide, polynucleotide or oligonucleotide can comprise the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil) and/or bases other than the five biologically occurring bases. These bases may serve a number of purposes, e.g., to stabilize or destabilize hybridization; to promote or inhibit probe degradation; or as attachment points for detectable moieties or quencher moieties.
  • a polynucleotide of the invention can contain one or more modified, non-standard, or derivatized base moieties, including, but not limited to, N 6 -methyl-adenine, N 6 -tert-butyl-benzyl-adenine, imidazole, substituted imidazoles, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methyl cytosine, 5-
  • 5-methoxyaminomethyl-2-thiouracil beta-D mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- methyluracil, uracil-5- oxyacetic acidmethylester, 3-(3-amino-3-N-2- carboxypropyl) uracil, (acp3)w, 2,6- diaminopurine, and 5-propynyl pyrimidine.
  • nucleic acid, nucleotide, polynucleotide or oligonucleotide can comprise one or more modified sugar moieties including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • a nucleic acid, nucleotide, polynucleotide or oligonucleotide can comprise phosphodiester linkages or modified linkages including, but not limited to phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages.
  • polynucleotide refers to a DNA, in particular cDNA, or RNA, in particular a cRNA, or a portion thereof. In the case of RNA (or cDN A), the polynucleotide is formed upon transcription of a nucleotide sequence that is capable of expression.
  • Polynucleotide fragments refer to fragments of between at least 8, such as 10, 12, 15 or 18 nucleotides and at least 50, such as 60, 80, 100, 200 or 300 nucleotides in length, or a complementary sequence thereto, e.g., representing a consecutive stretch of nucleotides of a gene, cDNA or mRNA.
  • polynucleotides also include any fragment (or complementary sequence thereto) of a sequence corresponding to or derived from any of the markers defined herein.
  • primer refers to an oligonucleotide having a hybridization specificity sufficient for the initiation of an enzymatic polymerization under predetermined conditions, for example in an amplification technique such as polymerase chain reaction (PCR), in a process of sequencing, in a method of reverse transcription and the like.
  • probe refers to an oligonucleotide having a hybridization specificity sufficient for binding to a defined target sequence under predetermined conditions, for example in an amplification technique such as a 5'-nuclease reaction, in a hybridization-dependent detection method, such as a Southern or
  • probes correspond at least in part to selected markers.
  • Primers and probes may be used in a variety of ways and may be defined by the specific use.
  • a probe can be immobilized on a solid support by any appropriate means, including, but not limited to: by covalent bonding, by adsorption, by hydrophobic and/or electrostatic interaction, or by direct synthesis on a solid support (see in particular patent application WO 92/10092).
  • a probe may be labeled by means of a label chosen, for example, from radioactive isotopes, enzymes, in particular enzymes capable of acting on a chromogenic, fluorescent or luminescent substrate (in particular a peroxidase or an alkaline phosphatase), chromophoric chemical compounds, chromogenic, fluorigenic or luminescent compounds, analogues of nucleotide bases, and ligands such as biotin.
  • a label chosen, for example, from radioactive isotopes, enzymes, in particular enzymes capable of acting on a chromogenic, fluorescent or luminescent substrate (in particular a peroxidase or an alkaline phosphatase), chromophoric chemical compounds, chromogenic, fluorigenic or luminescent compounds, analogues of nucleotide bases, and ligands such as biotin.
  • Illustrative fluorescent compounds include, for example, fluorescein, carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, Cy3, tetramethylrhodamine, Cy3.5, carboxy-x-rhodamine, Texas Red, Cy5, and Cy5.5.
  • Illustrative luminescent compounds include, for example, luciferin and 2,3- dihydrophthalazinediones, such as luminol. Other suitable labels are described herein or are otherwise known to those of skill in the art.
  • Oligonucleotides may be modified with chemical groups to enhance their performance or to facilitate the characterization of amplification products.
  • backbone-modified oligonucleotides such as those having phosphorothioate or methylphosphonate groups which render the oligonucleotides resistant to the nucleolytic activity of certain polymerases or to nuclease enzymes may allow the use of such enzymes in an amplification or other reaction.
  • Another example of modification involves using non-nucleotide linkers (e.g., Arnold, et al., "Non- Nucleotide Linking Reagents for Nucleotide Probes",
  • Amplification oligonucleotides may also contain mixtures of the desired modified and natural nucleotides.
  • a "reference" in the context of gene expression profiling refers to a cell and/or genes in or derived from the cell (or data derived therefrom) relative to which a target is compared. In some embodiments, for example, the expression of one or more genes from a target cell is compared to a corresponding expression of the genes in or derived from a reference cell.
  • sample refers to any biological material containing genetic information in the form of nucleic acids or proteins obtainable or obtained from one or more subjects or individuals.
  • samples are derived from subjects having leukemia, e.g., AML.
  • Exemplary samples include tissue samples, cell samples, bone marrow, and/or bodily fluids such as blood, saliva, semen, urine, and the like. Methods of obtaining samples and of isolating nucleic acids and proteins from sample are generally known to persons of skill in the art.
  • a “set” refers to a collection of one or more things. For example, a set may include 1 , 2, 3, 4, 5, 10, 20, 50, 100, 1 ,000 or another number of genes or other types of molecules.
  • a “solid support” refers to a solid material that can be derivatized with, or otherwise attached to, a chemical moiety, such as an oligonucleotide probe or the like.
  • Exemplary solid supports include plates (e.g., multi-well plates, etc.), beads, microbeads, tubes, fibers, whiskers, combs, hybridization chips (including microarray substrates, such as those used in GeneChip® probe arrays (Affymetrix, Inc., Santa Clara, CA, USA) and the like), membranes, single crystals, ceramic layers, self-assembling monolayers, and the like.
  • "Specifically binding” means that a compound is capable of discriminating between two or more polynucleotides or polypeptides.
  • the compound binds to the desired polynucleotide or polypeptide, but essentially does not bind to a non-target polynucleotide or polypeptide.
  • the compound can be an antibody, or a fragment thereof, an enzyme, a so-called small molecule compound, a protein- scaffold (e.g., an anticalin).
  • a "subject” refers to an organism.
  • the organism is a mammalian organism, particularly a human organism.
  • substantially identical in the context of gene expression refers to levels of expression of genes that are approximately equal to one another. In some embodiments, for example, the expression levels of genes being compared are substantially identical to one another when they differ by less than about 5% (e.g., about 4%, about 3%, about 2%, about 1%, etc.).
  • t(15;17) refers to AML with translocation t(15;17) according to the WHO classification of haematological malignancies.
  • t(8;21) refers to AML with translocation t(8;21) according to the WHO classification of haematological malignancies.
  • targets refers to an object that is the subject of analysis.
  • targets are specific nucleic acid sequences (e.g., mRNAs of expressed genes, etc.), the presence, absence or abundance of which are to be determined.
  • targets include polypeptides (e.g., proteins, etc.) of expressed genes.
  • sequences subjected to analysis are in or derived from "target cells", such as a particular type of leukemia cell.
  • RNA degradation RNA degradation, shipment time, or age of the patient can principally influence the measured gene expression. Accordingly, the present invention provides methods of validating gene expression assays.
  • the methods include (a) providing a population of cells and (b) subjecting at least two sub-populations of the cells to at least one different assay parameter (e.g., a duration of sample shipment time; a measure of RNA and/or protein quality; a duration of sample storage time prior to target preparation; a date of sample target preparation within a marker discovery study; an age of a subject at diagnosis; an assay operator; a washing condition; a detection condition; a specimen type; or the like).
  • at least one different assay parameter e.g., a duration of sample shipment time; a measure of RNA and/or protein quality; a duration of sample storage time prior to target preparation; a date of sample target preparation within a marker discovery study; an age of a subject at diagnosis; an assay operator; a washing condition; a detection condition; a specimen type; or the like.
  • the methods also include (c) detecting expression levels of substantially identical sets of genes in or derived from the cells in the sub-populations to produce at least two gene expression data sets and (d) determining whether the gene expression data sets are substantially similar to one another, thereby validating the diagnostic gene expression assay.
  • the methods include (e) selecting the gene expression data sets that are substantially similar to one another, and optionally (f) using the selected gene expression data sets in performing the diagnostic gene expression assay.
  • the methods include (e) repeating (b) - (d) using a modified assay parameter, if the gene expression data sets are not substantially similar to one another.
  • the method includes (a) subjecting at least two sub-populations of cells to at least one different assay parameter (b) detecting expression levels of substantially identical sets of genes in or derived from the cells in the sub-populations to produce at least two gene expression data sets and (c) determining whether the gene expression data sets are substantially similar to one another, thereby validating the diagnostic gene expression assay.
  • An example provided below illustrates that for a subset of leukemias expression profiling is applicable in a diagnostic setting considering various influencing parameters. More specifically, using a set of differentially expressed probes, four genetically defined AML subtypes with recurrent chromosomal aberrations can robustly be identified. In addition, preparations from different operators, and different sample handling procedures did not impair robustness of diagnostic expression signatures. In conclusion, this further highlights the applicability of microarrays and other gene expression analysis formats in a diagnostic setting. In practicing the present invention, many conventional techniques in, hematology, molecular biology and recombinant DNA are optionally used.
  • samples are collected and prepared for analysis using essentially any technique known to those of skill in the art.
  • samples are optionally obtained from essentially any source.
  • blood samples are obtained from subjects via venipuncture.
  • Whole blood specimens are optionally collected in EDTA, Heparin or ACD vacutainer tubes.
  • the samples utilized for analysis comprise bone marrow aspirates, which are optionally processed, e.g., by erythrocyte lysis techniques, Ficoll density gradient centrifugations, or the like.
  • Samples are typically either analyzed immediately following acquisition or stored frozen at, e.g., -80 0 C until being subjected to analysis. Sample collection and handling are also described in, e.g., Garland et al., Handbook of Phlebotomy and Patient Service Techniques, Lippincott Williams &
  • the cells lines or sources containing the target nucleic acids and/or expression products thereof are optionally subjected to one or more specific treatments that induce changes in gene expression, e.g., as part of processes to identify candidate modulators of gene expression.
  • a cell or cell line can be treated with or exposed to one or more chemical or biochemical constituents, e.g., pharmaceuticals, pollutants, DNA damaging agents, oxidative stress-inducing agents, pH-altering agents, membrane-disrupting agents, metabolic blocking agent, a chemical inhibitors, cell surface receptor ligands, antibodies, transcription promoters/enhancers/inhibitors, translation promoters/enhancers/inhibitors, protein- stabilizing or destabilizing agents, various toxins, carcinogens or teratogens, characterized or uncharacterized chemical libraries, proteins, lipids, or nucleic acids.
  • chemical or biochemical constituents e.g., pharmaceuticals, pollutants, DNA damaging agents, oxidative stress-inducing agents, pH-altering agents, membrane
  • the treatment comprises an environmental stress, such as a change in one or more environmental parameters including, but not limited to, temperature (e.g. heat shock or cold shock), humidity, oxygen concentration (e.g., hypoxia), radiation exposure, culture medium composition, or growth saturation.
  • environmental stress such as a change in one or more environmental parameters including, but not limited to, temperature (e.g. heat shock or cold shock), humidity, oxygen concentration (e.g., hypoxia), radiation exposure, culture medium composition, or growth saturation.
  • Responses to these treatments may be followed temporally, and the treatment can be imposed for various times and at various concentrations.
  • Target sequences can also be derived from cells exposed to multiple specific treatments as described above, either concurrently or in tandem (e.g., a cancerous cell or tissue sample may be further exposed to a DNA damaging agent while grown in an altered medium composition).
  • total RNA is isolated from samples for use as target sequences.
  • Cellular samples are lysed once culture with or without the treatment is complete by, for example, removing growth medium and adding a guanidinium- based lysis buffer containing several components to stabilize the RNA.
  • the lysis buffer also contains purified RNAs as controls to monitor recovery and stability of RNA from cell cultures. Examples of such purified RNA templates include the Kanamycin Positive Control RNA from Promega (Madison, WI, USA), and 7.5 kb Poly(A)-Tailed RNA from Life Technologies (Rockville, MD, USA). Lysates may be used immediately or stored frozen at, e.g., -80°C.
  • total RNA is purified from cell lysates (or other types of samples) using silica-based isolation in an automation-compatible, 96-well format, such as the Rneasy® purification platform (Qiagen, Inc. (Valencia, CA, USA)).
  • silica-based isolation in an automation-compatible, 96-well format, such as the Rneasy® purification platform (Qiagen, Inc. (Valencia, CA, USA)).
  • RNA is isolated using solid-phase oligo-dT capture using oligo-dT bound to microbeads or cellulose columns. This method has the added advantage of isolating mRNA from genomic DNA and total RNA, and allowing transfer of the mRNA-capture medium directly into the reverse transcriptase reaction.
  • Other RNA isolation methods are contemplated, such as extraction with silica-coated beads or guanidinium. Further methods for RNA isolation and preparation can be devised by one skilled in the art.
  • the methods of the present invention are performed using crude cell lysates, eliminating the need to isolate RNA.
  • RNAse inhibitors are optionally added to the crude samples.
  • genomic DNA could contribute one or more copies of target sequence, depending on the sample.
  • the signal arising from genomic DNA may not be significant. But for genes expressed at very low levels, the background can be eliminated by treating the samples with DNAse, or by using primers that target splice junctions.
  • the determination of gene expression levels may be effected at the transcriptional and/or translational level, i.e., at the level of mRNA or at the protein level.
  • any method of gene expression profiling can be used or adapted for use in performing the methods described herein including, e.g., methods based on hybridization analysis of polynucleotides, and methods based on sequencing of polynucleotides.
  • methods for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization
  • RNAse protection assays Hod, Biotechniques 13:852-854 (1992)
  • RT-PCR reverse transcription polymerase chain reaction
  • antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
  • Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS).
  • molecular species such as antibodies, aptamers, etc.
  • the methods described herein include determining the expression levels of transcribed polynucleotides.
  • the transcribed polynucleotide is an mRNA, a cDNA and/or a cRNA. Transcribed polynucleotides are typically isolated from a sample, reverse transcribed and/or amplified, and labeled by techniques referred to above or otherwise known to persons skilled in the art.
  • the methods of the invention generally include hybridizing transcribed polynucleotides to a complementary polynucleotide, or a portion thereof, under a selected hybridization condition (e.g., a stringent hybridization condition), as described herein.
  • a selected hybridization condition e.g., a stringent hybridization condition
  • the detection and quantification of amounts of polynucleotides to determine the level of expression of a marker are performed according to those described by, e.g., Sambrook et al., supra, or real time methods known in the art as 5'-nuclease methods disclosed in, e.g., WO 92/02638, U.S. Pat.
  • 5 '-nuclease methods utilize the exonuclease activity of certain polymerases to generate signals.
  • target nucleic acids are detected in processes that include contacting a sample with an oligonucleotide containing a sequence complementary to a region of the target nucleic acid component and a labeled oligonucleotide containing a sequence complementary to a second region of the same target nucleic acid component sequence strand, but not including the nucleic acid sequence defined by the first oligonucleotide, to create a mixture of duplexes during hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the first oligonucleotide and to the labeled oligonucleotide such that the
  • 3 '-end of the first oligonucleotide is adjacent to the 5'-end of the labeled oligonucleotide. Then this mixture is treated with a template-dependent nucleic acid polymerase having a 5' to 3' nuclease activity under conditions sufficient to permit the to 3' nuclease activity of the polymerase to cleave the annealed, labeled oligonucleotide and release labeled fragments. The signal generated by the hydrolysis of the labeled oligonucleotide is detected and/or measured. 5'-nuclease technology eliminates the need for a solid phase bound reaction complex to be formed and made detectable.
  • exemplary methods include, e.g., fluorescence resonance energy transfer between two adjacently hybridized probes as used in the LightCycler® format described in, e.g., U.S. Pat. No. 6,174,670, which is incorporated by reference.
  • the marker i.e., the polynucleotide
  • the marker is in form of a transcribed nucleotide, where total RNA is isolated, cDNA and, subsequently, cRNA is synthesized and biotin is incorporated during the transcription reaction.
  • the purified cRNA is applied to commercially available arrays that can be obtained from, e.g., Affymetrix, Inc. (Santa Clara, CA USA).
  • the hybridized cRNA is optionally detected according to the methods described in the examples provided below.
  • the arrays are produced by photolithography or other methods known to persons skilled in the art. Some of these techniques are also described in, e.g. U.S. Pat. No. 5,445,934, U.S. Pat. No. 5,744,305, U.S. Pat. No. 5,700,637, U.S. Pat. No.
  • the polynucleotide or at least one of the polynucleotides is in form of a polypeptide (e.g., expressed from the corresponding polynucleotide).
  • the expression level of the polynucleotides or polypeptides is optionally detected using a compound that specifically binds to target polynucleotides or target polypeptides.
  • Some of the earliest expression profiling methods are based on the detection of a label in RNA hybrids or protection of RNA from enzymatic degradation (see, e.g., Ausubel et al., supra).
  • Methods based on detecting hybrids include northern blots and slot/dot blots. These two techniques differ in that the components of the sample being analyzed are resolved by size in a northern blot prior to detection, which enables identification of more than one species simultaneously.
  • Slot blots are generally carried out using unresolved mixtures or sequences, but can be easily performed in serial dilution, enabling a more quantitative analysis.
  • In situ hybridization is a technique that monitors transcription by directly visualizing RNA hybrids in the context of a whole cell. This method provides information regarding subcellular localization of transcripts (see, e.g., Suzuki et al., Pigment Cell Res. 17(l):10-4 (2004)).
  • RNAse protection assays employ a labeled nucleic acid probe, which is hybridized to the RNA species being analyzed, followed by enzymatic degradation of single-stranded regions of the probe. Analysis of the amount and length of probe protected from degradation is used to determine the quantity and endpoints of the transcripts being analyzed.
  • RT-PCR Reverse Transcriptase PCR
  • RT-PCR can be used to compare, e.g., mRNA levels in different sample populations, in normal and tumor tissues, with or without drug treatment, to characterize patterns of gene expression, to discriminate between closely related mRNAs, and to analyze RNA structure.
  • assays are derivatives of PCR in which amplification is preceded by reverse transcription of mRNA into cDNA. Accordingly, an initial step in these processes is generally the isolation of mRNA from a target sample (e.g., leukemia cells).
  • the starting material is typically total RNA isolated from cancerous tissues or cells (e.g., bone marrow, peripheral blood aliquots, etc.), and in certain embodiments, from corresponding normal tissues or cells.
  • RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions.
  • total RNA from cells in culture can be isolated using Qiagen Rneasy® mini-columns (referred to above).
  • Other commercially available RNA isolation kits include MasterPureTM Complete DNA and RNA Purification Kit (EPICENTRETM, Madison, Wis.), and Paraffin Block RNA Isolation Kit (Ambion, Inc.).
  • Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test).
  • RNA prepared from tumor can be isolated, for example, by cesium chloride density gradient centrifugation.
  • RNA generally cannot serve as a template for PCR
  • the process of gene expression profiling by RT-PCR typically includes the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction.
  • Two commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT).
  • AMV-RT avilo myeloblastosis virus reverse transcriptase
  • MMLV-RT Moloney murine leukemia virus reverse transcriptase
  • the reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the particular circumstances of expression profiling analysis.
  • extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, CA, USA), following the manufacturer's instructions.
  • the derived cDNA can then be used as a template in the subsequent PCR reaction.
  • the PCR step can use a variety of thermostable DNA-dependent DNA polymerases, it typically employs the Taq DNA polymerase, which has a 5 '-3' nuclease activity but lacks a 3'-5' proofreading endonuclease activity.
  • TaqMan® PCR typically utilizes the 5'-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used. Pairs of primers are generally used to generate amplicons in PCR reactions.
  • a third oligonucleotide, or probe is designed to bind to nucleotide sequence located between PCR primer pairs.
  • Probe are generally non-extendible by Taq DNA polymerase enzyme, and are typically labeled with, e.g., a reporter fluorescent dye and a quencher fluorescent dye.
  • Laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together, such as in an intact probe.
  • the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner.
  • the resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore.
  • One molecule of reporter dye is typically liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.
  • TaqMan® RT-PCR can be performed using commercially available equipment, such as, for example, a LightCycler® system (Roche Molecular Biochemicals,
  • RT-PCR is typically performed using an internal standard.
  • An ideal internal standard is expressed at a relatively constant level among different cells or tissues, and is unaffected by the experimental treatment.
  • Exemplary RNAs frequently used to normalize patterns of gene expression are mRNAs transcribed from for the housekeeping genes glyceraldehyde-3-phosphate-dehydrogenase (GAPD) and ⁇ - actin.
  • Other exemplary methods for targeted mRNA analysis include differential display reverse transcriptase PCR (DDRT-PCR) and RNA arbitrarily primed PCR (RAP- PCR) (see, e.g., U.S. Patent No.
  • Molecular beacons are oligonucleotides designed for real time detection and quantification of target nucleic acids.
  • the 5' and 3' termini of molecular beacons collectively comprise a pair of moieties, which confers the detectable properties of the molecular beacon.
  • One of the termini is attached to a fluorophore and the other is attached to a quencher molecule capable of quenching a fluorescent emission of the fluorophore.
  • a fluorophore-quencher pair can use a fluorophore, such as EDANS or fluorescein, e.g., on the 5'-end and a quencher, such as Dabcyl, e.g., on the 3'-end.
  • the stem of the molecular beacon is stabilized by complementary base pairing.
  • This self-complementary pairing results in a "hairpin loop" structure for the molecular beacon in which the fluorophore and the quenching moieties are proximal to one another. In this confirmation, the fluorescent moiety is quenched by the quenching moiety.
  • the loop of the molecular beacon typically comprises the oligonucleotide probe and is accordingly complementary to a sequence to be detected in the target microbial nucleic acid, such that hybridization of the loop to its complementary sequence in the target forces disassociation of the stem, thereby distancing the fluorophore and quencher from each other. This results in unquenching of the fluorophore, causing an increase in fluorescence of the molecular beacon. Details regarding standard methods of making and using molecular beacons are well established in the literature and molecular beacons are available from a number of commercial reagent sources. Further details regarding methods of molecular beacon manufacture and use are found, e.g., in Leone et al.
  • kits which utilize molecular beacons are also commercially available, such as the SentinelTM Molecular Beacon Allelic
  • oligonucleotides e.g., microarrays
  • polynucleotide sequences of interest e.g., probes, such as cDNAs, mRNAs, oligonucleotides, etc.
  • probes such as cDNAs, mRNAs, oligonucleotides, etc.
  • microchip substrate or other type of solid support
  • Sequences of interest can be obtained, e.g., by creating a cDNA library from an mRNA source or by using publicly available databases, such as GenBank, to annotate the sequence information of custom cDNA libraries or to identify cDNA clones from previously prepared libraries.
  • the arrayed sequences are then hybridized with target nucleic acids from cells or tissues of interest.
  • the source of mRNA typically is total RNA isolated from a sample.
  • high-density oligonucleotide arrays are produced using a light-directed chemical synthesis process (i.e., photolithography). Unlike common cDNA arrays, oligonucleotide arrays (according, e.g., to the Affymetrix technology) typically use a single-dye technology. Given the sequence information of the probes or markers, the sequences are typically synthesized directly onto the array, thus, bypassing the need for physical intermediates, such as PCR products, commonly utilized in making cDNA arrays.
  • markers, or partial sequences thereof can be represented by, e.g., between about 14 to 20 features, typically by less then 14 features, more typically less then about 10 features, even more typically by about 6 features or less, with each feature generally being a short sequence of nucleotides (oligonucleotide), which is typically a perfect match (PM) to a segment of the respective gene.
  • oligonucleotide typically a perfect match (PM) to a segment of the respective gene.
  • the PM oligonucleotides are paired with mismatch (MM) oligonucleotides, which have a single mismatch at the central base of the nucleotide and are used as "controls".
  • the chip exposure sites are typically defined by masks and are de-protected by the use of light, followed by a chemical coupling step resulting in the synthesis of one nucleotide.
  • the masking, light deprotection, and coupling process can then be repeated to synthesize the next nucleotide, until the nucleotide chain is of the specified length.
  • PCR amplified inserts of cDNA clones are applied to a substrate in a dense array.
  • at least 10,000 different cDNA probe sequences are applied to a given solid support.
  • Fluorescently labeled cDNA targets may be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from the samples of interest.
  • Labeled cDNA targets applied to the chip hybridize with corresponding probes on the array. After washing (e.g., under stringent conditions) to remove non-specifically bound probes, the chip is typically scanned by confocal laser microscopy or by another detection method, such as a CCD camera.
  • Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance.
  • dual color fluorescence for example, separately labeled cDNA probes generated from two sources of RNA can be hybridized concurrently to the arrayed probes.
  • the relative abundance of the transcripts from the two sources corresponding to each specified gene can thus be determined simultaneously.
  • the miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes.
  • Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et al., Proc. Natl. Acad. Sci.
  • microarray-based assay formats are also optionally utilized. Microarray analysis can be performed using commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GeneChip® technology, or Agilent's microarray technology. If the polynucleotide being detected is mRNA, cDNA may be prepared into which a detectable label, as exemplified herein, is incorporated.
  • labeled cDNA in single-stranded form, may then be hybridized (e.g., under stringent or highly stringent conditions) to a panel of single-stranded oligonucleotides representing different genes and affixed to a solid support, such as a chip.
  • a solid support such as a chip.
  • those cDNAs that have a counterpart in the oligonucleotide panel or array will be detected (e.g., quantitatively detected).
  • mRNA or cDNA may be amplified, e.g., by a polymerase chain reaction or another nucleic acid amplification technique.
  • the number of amplified copies corresponds to the number of mRNAs originally present in the cell.
  • cDNAs are transcribed into cRNAs prior to hybridization steps in a given assay.
  • labels can be attached or incorporated cRNAs during or after the transcription step.
  • one exemplary embodiment of the methods of the invention includes, as follows (1) obtaining a sample, e.g.
  • RNA e.g., mRNA
  • RNA e.g., mRNA
  • reverse transcribing the RNA into cDNA e.g., RNA into cDNA
  • in vitro transcribing the cDNA into cRNA e.g., the HG-Ul 33 microarray set available from Affymetrix, Inc. (Santa Clara, CA USA)
  • fragmented cRNA e.g., the HG-Ul 33 microarray set available from Affymetrix, Inc. (Santa Clara, CA USA)
  • detecting hybridization e.g., the HG-Ul 33 microarray set available from Affymetrix, Inc. (Santa Clara, CA USA)
  • Serial analysis of gene expression is a method that allows the simultaneous and quantitative analysis of a large number of gene transcripts, without the need for providing an individual hybridization probe for each transcript.
  • a short sequence tag e.g., about 10-14 bp
  • many transcripts are linked together to form long serial molecules, that can be sequenced, revealing the identity of the multiple tags simultaneously.
  • the expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags, and identifying the gene corresponding to each tag.
  • SAGE-based assays are also described in, e.g. Velculescu et al., Science 270:484- 487 (1995) and Velculescu et al., Cell 88:243-51 (1997), which are both incorporated by reference.
  • a microbead library of DNA templates is constructed by in vitro cloning. This is generally followed by the assembly of a planar array of the template-containing microbeads in a flow cell at a high density (typically greater than 3 x 10 6 microbeads/cm 2 ). The free ends of the cloned templates on each microbead are analyzed simultaneously, using a fluorescence- based signature sequencing method that does not require DNA fragment separation. This method can be used to simultaneously and accurately provide, in a single operation, hundreds of thousands of gene signature sequences from cDNA libraries. MPSS is also described in, e.g., Brenner et al., (2000) Nature Biotechnology 18:630-634, which is incorporated by reference.
  • any available technique for the detection of proteins is optionally utilized in the methods of the invention.
  • Exemplary protein analysis technologies include, e.g., one- and two-dimensional SDS-P AGE-based separation and detection, immunoassays (e.g., western blotting, etc.), aptamer-based detection, mass spectrometric detection, and the like. These and other techniques are generally well-known in the art.
  • immunohistochemical methods are optionally used for detecting the expression levels of the targets described herein.
  • antibodies or antisera e.g., polyclonal antisera
  • monoclonal antibodies specific for particular targets are used to detect expression.
  • antibodies are directly labeled, e.g., with radioactive labels, fluorescent labels, haptens, chemiluminescent dyes, enzyme substrates or co-factors, enzyme inhibitors, free radicals, enzymes (e.g., horseradish peroxidase or alkaline phosphatase), or the like.
  • labeled reagents may be used in a variety of well known assays, such as radioimmunoassays, enzyme immunoassays, e.g., ELlSA, fluorescent immunoassays, and the like. See, e.g., U.S. Pat. Nos. 3,766,162; 3,791,932; 3,817,837; and 4,233,402, which are each incorporated by reference.
  • unlabeled primary antibodies are used in conjunction with labeled secondary antibodies, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody.
  • Immunohistochemistry protocols and kits are well known in the art and are commercially available.
  • proteins from a cell or tissue under investigation may be contacted with a panel or array of aptamers or of antibodies or fragments or derivatives thereof. These biomolecules may be affixed to a solid support, such as a chip.
  • the binding of proteins indicative of a given leukemia type or subtype is optionally verified by binding to a detectably labeled secondary antibody or aptamer.
  • the labeling of antibodies is also described in, e.g., Harlow and Lane, Antibodies, a laboratory manual, CSH Press (1988), which is incorporated by reference.
  • a minimum set of proteins necessary for detecting various leukemia types or subtypes may be selected for the creation of a protein array for use in making diagnoses with, e.g., protein lysates of bone marrow samples directly.
  • Protein array systems for the detection of specific protein expression profiles are commercially available from various suppliers, including the Bio-PlexTM platform available from BIO-RAD Laboratories (Munich, Germany).
  • antibodies against the target proteins are produced and immobilized on a solid support, e.g., a glass slide or a well of a microtiter plate.
  • the immobilized antibodies can be labeled with a reactant that is specific for the target proteins.
  • reactants can include, e.g., enzyme substrates, DNA, receptors, antigens or antibodies to create for example a capture sandwich immunoassay.
  • Target proteins can also be detected using aptamers including photoaptamers.
  • Aptamers generally are single-stranded oligonucleotides (e.g., typically DNA for diagnostic applications) that assume a specific, sequence-dependent shape and binds to target proteins based on a "lock-and-key" fit between the two molecules.
  • the detection of proteins via mass includes various formats that can be adapted for use in the methods of the invention.
  • Exemplary formats include matrix assisted laser desorption/ionization- (MALDI) and surface enhanced laser desorption/ionization-based (SELDl) detection.
  • MALDI- and SELDI-based detection are also described in, e.g., Weinberger et al. (2000) "Recent trends in protein biochip technology," Pharmacogenomics 1(4):395-416, Forde et al. (2002) “Characterization of transcription factors by mass spectrometry and the role of SELDI-MS,” Mass Spectrom. Rev. 21(6):419-439, and Leushner (2001) "MALDI TOF mass spectrometry: an emerging platform for genomics and diagnostics,"
  • oligonucleotides for use as probes and/or primers.
  • the DNAstar software package available from DNASTAR, Inc. can be used for sequence alignments.
  • target nucleic acid sequences and non-target nucleic acid sequences can be uploaded into DNAstar EditSeq program as individual files, e.g., as part of a process to identify regions in these sequences that have low sequence similarity.
  • pairs of sequence files can be opened in the DNAstar MegAlign sequence alignment program and the Clustal W method of alignment can be applied.
  • the parameters used for Clustal W alignments are optionally the default settings in the software.
  • MegAlign typically does not provide a summary of the percent identity between two sequences. This is generally calculated manually. From the alignments, regions having, e.g., less than
  • sequence alignment algorithms are also described in, e.g., Mount, Bioinformatics: Sequence and Genome Analysis, Cold Spring Harbor Laboratory Press (2001 ), and Durbin et al.,
  • optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman &
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle (1987) J. MoI. Evol. 35:351-360, which is incorporated by reference.
  • Oligonucleotide probes and primers are optionally prepared using essentially any technique known in the art.
  • the oligonucleotide probes and primers are synthesized chemically using essentially any nucleic acid synthesis method, including, e.g., according to the solid phase phosphoramidite method described by Beaucage and Caruthers (1981) Tetrahedron Letts. 22(20): 1859-1862, which is incorporated by reference.
  • oligonucleotides can also be synthesized using a triester method (see, e.g., Capaldi et al.
  • primer nucleic acids optionally include various modifications.
  • primers include restriction site linkers, e.g., to facilitate subsequent amplicon cloning or the like.
  • primers are also optionally modified to improve the specificity of amplification reactions as described in, e.g., U.S. Pat. No.
  • Probes and/or primers utilized in the methods and other aspects of the invention are typically labeled to permit detection of probe-target hybridization duplexes.
  • a label can be any moiety that can be attached to a nucleic acid and provide a detectable signal (e.g., a quantifiable signal).
  • Labels may be attached to oligonucleotides directly or indirectly by a variety of techniques known in the art. To illustrate, depending on the type of label used, the label can be attached to a terminal (5' or 3' end of an oligonucleotide primer and/or probe) or a non-terminal nucleotide, and can be attached indirectly through linkers or spacer arms of various sizes and compositions.
  • oligonucleotides containing functional groups e.g., thiols or primary amines
  • functional groups e.g., thiols or primary amines
  • oligonucleotides can label such oligonucleotides using protocols described in, e.g., Innis et al. (Eds.) PCR Protocols: A Guide to Methods and Applications, Elsevier Science & Technology Books (1990)(Innis), which is incorporated by reference.
  • labels comprise a fluorescent dye (e.g., a rhodamine dye (e.g., R6G, Rl 10, TAMRA, ROX, etc.), a fluorescein dye (e.g., JOE, VIC, TET, HEX, FAM, etc.), a halofluorescein dye, a cyanine dye (e.g., CY3, CY3.5, CY5, CY5.5, etc.), a BODIPY® dye (e.g., FL, 530/550, TR, TMR, etc.), an ALEXA FLUOR® dye (e.g., 488, 532, 546, 568, 594, 555, 653, 647, 660, 680, etc.), a dichlororhodamine dye, an energy transfer dye (e.g., BIGDYETM
  • Fluorescent dyes are generally readily available from various commercial suppliers including, e.g., Molecular Probes, Inc. (Eugene, OR), Amersham Biosciences Corp. (Piscataway, NJ), Applied Biosystems (Foster City,
  • labels include, e.g., biotin, weakly fluorescent labels (Yin et al. (2003) Appl Environ Microbiol. 69(7):3938, Babendure et al. (2003) Anal. Biochem. 317(1):!. and Jankowiak et al. (2003) Chem Res Toxicol. 16(3):304), non-fluorescent labels, colorimetric labels, chemiluminescent labels (Wilson et al. (2003) Analyst. 128(5):480 and Roda et al. (2003) Luminescence 18(2):72), Raman labels, electrochemical labels, bioluminescent labels (Kitayama et al. (2003) Photochem Photobiol.
  • nucleic acid labeling is also described further below.
  • labeling is achieved using synthetic nucleotides (e.g., synthetic ribonucleotides, etc.) and/or recombinant phycoerythrin (PE).
  • a fluorescent dye is a label or a quencher is generally defined by its excitation and emission spectra, and the fluorescent dye with which it is paired.
  • Fluorescent molecules commonly used as quencher moieties in probes and primers include, e.g., fluorescein, FAM, JOE, rhodamine, R6G, TAMRA, ROX, DABCYL, and EDANS. Many of these compounds are available from the commercial suppliers referred to above.
  • Exemplary non-fluorescent or dark quenchers that dissipate energy absorbed from a fluorescent dye include the Black
  • Hole QuenchersTM or BHQTM which are commercially available from Biosearch Technologies, Inc. (Novato, CA, USA).
  • nucleic acid can be custom or standard ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company, The Great American Gene Company, ExpressGen Inc., Operon Technologies Inc., Proligo LLC, and many others.
  • modified nucleotides are included in probes and primers.
  • the introduction of modified nucleotide substitutions into oligonucleotide sequences can, e.g., increase the melting temperature of the oligonucleotides. In some embodiments, this can yield greater sensitivity relative to corresponding unmodified oligonucleotides even in the presence of one or more mismatches in sequence between the target nucleic acid and the particular oligonucleotide.
  • modified nucleotides that can be substituted or added in oligonucleotides include, e.g., C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6- diaminopurines, C5-propynyl-dC, C7-propynyl-dA, C7-propynyl-dG, C5- propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA, C7- propargylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidine analogs, pseudo-dU, nitro pyrrole, nitro indole, 2'-0-methyl Ribo-U, 2'-0-methyl Ribo-C, an 8-aza-dA, an 8-aza-dG, a 7-deaza-dA, a 7-d-
  • modified oligonucleotides include those having one or more LNATM monomers.
  • Nucleotide analogs such as these are also described in, e.g., U.S. Pat. No. 6,639,059, entitled “SYNTHESIS OF [2.2.I]BlCYCLO NUCLEOSIDES,” issued October 28, 2003 to Kochkine et al., U.S. Pat. No. 6,303,315, entitled "ONE STEP SAMPLE PREPARATION AND
  • oligonucleotide probes designed to hybridize with target nucleic acids are covalently or noncovalently attached to solid supports.
  • labeled amplicons derived from patient samples are typically contacted with these solid support-bound probes to effect hybridization and detection.
  • amplicons are attached to solid supports and contacted with labeled probes.
  • antibodies, aptamers, or other probe biomolecules utilized in a given assay are similarly attached to solid supports.
  • any substrate material can be adapted for use as a solid support.
  • substrates are fabricated from silicon, glass, or polymeric materials (e.g., glass or polymeric microscope slides, silicon wafers, wells of microwell plates, etc.).
  • Suitable glass or polymeric substrates, including microscope slides, are available from various commercial suppliers, such as Fisher Scientific (Pittsburgh, PA, USA) or the like.
  • solid supports utilized in the invention are membranes. Suitable membrane materials are optionally selected from, e.g.
  • polyaramide membranes polycarbonate membranes, porous plastic matrix membranes (e.g., POREX® Porous Plastic, etc.), nylon membranes, ceramic membranes, polyester membranes, polytetrafluoroethylene (TEFLON®) membranes, nitrocellulose membranes, or the like.
  • POREX® Porous Plastic porous plastic matrix membranes
  • nylon membranes e.g
  • exemplary solid supports that are optionally utilized include, e.g., ceramics, metals, resins, gels, plates, beads (e.g., magnetic microbeads, etc.), whiskers, fibers, combs, single crystals, self- assembling monolayers, and the like.
  • Nucleic acids are directly or indirectly (e.g., via linkers, such as bovine serum albumin (BSA) or the like) attached to the supports, e.g., by any available chemical or physical method.
  • linkers such as bovine serum albumin (BSA) or the like
  • a wide variety of linking chemistries are available for linking molecules to a wide variety of solid supports. More specifically, nucleic acids may be attached to the solid support by covalent binding, such as by conjugation with a coupling agent or by non-covalent binding, such as electrostatic interactions, hydrogen bonds or antibody-antigen coupling, or by combinations thereof.
  • Typical coupling agents include biotin/avidin, biotin/streptavidin, Staphylococcus aureus protein A/IgG antibody F c fragment, and streptavidin/protein A chimeras (Sano et al. (1991) Bio/Technology 9:1378, which is incorporated by reference), or derivatives or combinations of these agents.
  • Nucleic acids may be attached to the solid support by a photocleavable bond, an electrostatic bond, a disulfide bond, a peptide bond, a diester bond or a combination of these bonds. Nucleic acids are also optionally attached to solid supports by a selectively releasable bond such as 4,4'-dimethoxytrityl or its derivative.
  • Cleavable attachments can be created by attaching cleavable chemical moieties between the probes and the solid support including, e.g., an oligopeptide, oligonucleotide, oligopolyamide, oligoacrylamide, oligoethylene glycerol, alkyl chains of between about 6 to 20 carbon atoms, and combinations thereof. These moieties may be cleaved with, e.g., added chemical agents, electromagnetic radiation, or enzymes.
  • Exemplary attachments cleavable by enzymes include peptide bonds, which can be cleaved by proteases, and phosphodiester bonds which can be cleaved by nucleases.
  • Chemical agents such as ⁇ -mercaptoethanol, dithiothreitol (DTT) and other reducing agents cleave disulfide bonds.
  • Other agents which may be useful include oxidizing agents, hydrating agents and other selectively active compounds.
  • Electromagnetic radiation such as ultraviolet, infrared and visible light cleave photocleavable bonds. Attachments may also be reversible, e.g., using heat or enzymatic treatment, or reversible chemical or magnetic attachments. Release and reattachment can be performed using, e.g., magnetic or electrical fields.
  • the length of complementary region or sequence between primer or probes and their binding partners should generally be sufficient to allow selective or specific hybridization of the primers or probes to the targets at the selected annealing temperatures used for a particular nucleic acid amplification protocol, expression profiling assay, etc.
  • complementary regions of, for example, between about 10 and about 50 nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 or more nucleotides) are typically used in a given application.
  • “Stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments, such as Southern and northern hybridizations, are sequence dependent, and are different under different environmental parameters.
  • highly stringent hybridization and wash conditions are selected to be about 5° C or less lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH (as noted below, highly stringent conditions can also be referred to in comparative terms).
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the test sequence hybridizes to a perfectly matched primer or probe.
  • Very stringent conditions are selected to be equal to the T 111 for a particular primer or probe.
  • the T m is the temperature of the nucleic acid duplexes indicates the temperature at which the duplex is 50% denatured under the given conditions and its represents a direct measure of the stability of the nucleic acid hybrid.
  • the T m corresponds to the temperature corresponding to the midpoint in transition from helix to random coil; it depends on length, nucleotide composition, and ionic strength for long stretches of nucleotides.
  • unhybridized nucleic acid material can be removed by a series of washes, the stringency of which can be adjusted depending upon the desired results. Low stringency washing conditions (e.g., using higher salt and lower temperature) increase sensitivity, but can product nonspecific hybridization signals and high background signals.
  • one measure of stringent hybridization is the ability of the primer or probe to hybridize to one or more of the target nucleic acids (or complementary polynucleotide sequences thereof) under highly stringent conditions. Stringent hybridization and wash conditions can easily be determined empirically for any test nucleic acid.
  • the hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and/or increasing the concentration of organic solvents, such as formalin, in the hybridization or wash), until a selected set of criteria is met.
  • the hybridization and wash conditions are gradually increased until a target nucleic acid, and complementary polynucleotide sequences thereof, binds to a perfectly matched complementary nucleic acid.
  • a target nucleic acid is said to specifically hybridize to a primer or probe nucleic acid when it hybridizes at least Vi as well to the primer or probe as to a perfectly matched complementary target, i.e., with a signal to noise ratio at least 1 A as high as hybridization of the primer or probe to the target under conditions in which the perfectly matched primer or probe binds to the perfectly matched complementary target with a signal to noise ratio that is at least about 2.5x-10x, typically 5x-10x as high as that observed for hybridization to any of the unmatched target nucleic acids.
  • RNA is converted to cDNA in a reverse-transcription (RT) reaction using, e.g., a target-specific primer complementary to the RNA for each gene target being monitored.
  • RT reverse-transcription
  • Methods of reverse transcribing RNA into cDNA are well known, and described in Sambrook, supra.
  • Alternative methods for reverse transcription utilize thermostable DNA polymerases, as described in the art.
  • avian myeloblastosis virus reverse transcriptase (AMV- RT), or Maloney murine leukemia virus reverse transcriptase (MoMLV-RT) is used, although other enzymes are also optionally utilized.
  • AMV- RT avian myeloblastosis virus reverse transcriptase
  • MoMLV-RT Maloney murine leukemia virus reverse transcriptase
  • An advantage of using target-specific primers in the RT reaction is that only the desired sequences are converted into a PCR template.
  • RNA targets are reverse transcribed using non-specific primers, such as an anchored oligo-dT primer, or random sequence primers.
  • non-specific primers such as an anchored oligo-dT primer, or random sequence primers.
  • An advantage of this embodiment is that the "unfractionated" quality of the mRNA sample is maintained because the sites of priming are non-specific, i.e., the products of this RT reaction will serve as template for any desired target in the subsequent PCR amplification. This allows samples to be archived in the form of DNA, which is more stable than RNA.
  • transcription-based amplification systems are used, such as that first described by Kwoh et al. (Proc. Natl. Acad. Sci. (1989)
  • mRNA target of interest is copied into cDNA by a reverse transcriptase.
  • the primer for cDNA synthesis includes the promoter sequence of a designated DNA-dependent RNA polymerase 5' to the primer's region of homology with the template.
  • RNA polymerase RNA polymerase
  • Transcription of the cDNA template rapidly amplifies the signal from the original target mRNA.
  • the isothermal reactions bypass the need for denaturing cDNA strands from their RNA templates by including RNAse H to degrade RNA hybridized to DNA.
  • amplification is accomplished by used of the ligase chain reaction (LCR), disclosed in European Patent Application No. 320,308
  • LDR ligase detection reaction
  • U.S. Patent No. 4,883,750 Whiteley et al.
  • two probe pairs are typically prepared, which are complimentary each other, and to adjacent sequences on both strands of the target. Each pair will bind to opposite strands of the target such that they abut. Each of the two probe pairs can then be linked to form a single unit, using a thermostable ligase. By temperature cycling, as in PCR, bound ligated units dissociate from the target, then both molecules can serve as "target sequences" for ligation of excess probe pairs, providing for an exponential amplification.
  • the LDR is very similar to LCR.
  • oligonucleotides complimentary to only one strand of the target are used, resulting in a linear amplification of ligation products, since only the original target DNA can serve as a hybridization template. It is used following a PCR amplification of the target in order to increase signal.
  • strand displacement amplification (Walker et al. (1992) Nucleic Acids Res. 20:1691-1696), repair chain reaction (REF), cyclic probe reaction (REF), solid-phase amplification, including bridge amplification (Mehta and Singh ( 1999) BioTechniques 26(6): 1082-1086), rolling circle amplification (Kool, U.S. Patent No. 5,714,320), rapid amplification of cDNA ends (Frohman (1988) Proc. Natl. Acad. Sci. 85: 8998-9002), and the "invader assay” (Griffin et al. (1999) Proc. Natl. Acad. Sci. 96: 6301-6306), which are each incorporated by reference.
  • Amplicons are optionally recovered and purified from other reaction components by any of a number of methods well known in the art, including electrophoresis, chromatography, precipitation, dialysis, filtration, and/or centrifugation. Aspects of nucleic acid purification are described in, e.g., Douglas et al., DNA Chromatography, Wiley, John & Sons, Inc. (2002), and Schott, Affinity
  • amplicons are not purified prior to detection, such as when amplicons are detected simultaneous with amplification.
  • the number of species than can be detected within a mixture depends primarily on the resolution capabilities of the separation platform used, and the detection methodology employed. In some embodiments, separation steps are is based upon size-based separation technologies. Once separated, individual species are detected and quantitated by either inherent physical characteristics of the molecules themselves, or detection of an associated label.
  • Embodiments employing other separation methods are also described.
  • certain types of labels allow resolution of two species of the same mass through deconvolution of the data.
  • Non-size based differentiation methods allow pooling of a plurality of multiplexed reactions to further increase throughput.
  • Certain embodiments of the invention incorporate a step of separating the products of a reaction based on their size differences.
  • the PCR products generated during an amplification reaction typically range from about 50 to about 500 bases in length, which can be resolved from one another by size.
  • Any one of several devices may be used for size separation, including mass spectrometry, any of several electrophoretic devices, including capillary, polyacrylamide gel, or agarose gel electrophoresis, or any of several chromatographic devices, including column chromatography, HPLC, or FPLC.
  • sample analysis includes the use of mass spectrometry.
  • mass spectrometry Several modes of separation that determine mass are possible, including Time-of- Flight (TOF), Fourier Transform Mass Spectrometry (FTMS), and quadruple mass .
  • TOF Time-of- Flight
  • FTMS Fourier Transform Mass Spectrometry
  • spectrometry Possible methods of ionization include Matrix-Assisted Laser Desorption and Ionization (MALDI) or Electrospray Ionization (ESI).
  • MALDI-TOF Wang, et al. (1993) Rapid Communications in Mass Spectrometry 7:142-146, which is incorporated by reference). This method may be used to provide unfragmented mass spectra of mixed-base oligonucleotides containing between about 1 and about 1000 bases.
  • the analyte is mixed into a matrix of molecules that resonantly absorb light at a specified wavelength. Pulsed laser light is then used to desorb oligonucleotide molecules out of the absorbing solid matrix, creating free, charged oligomers and minimizing fragmentation.
  • An exemplary solid matrix material for this purpose is 3-hydroxypicolinic acid (Wu, supra), although others are also optionally used.
  • a microcapillary is used for analysis of nucleic acids obtained from the sample.
  • Microcapillary electrophoresis generally involves the use of a thin capillary or channel, which may optionally be filled with a particular medium to improve separation, and employs an electric field to separate components of the mixture as the sample travels through the capillary.
  • Samples composed of linear polymers of a fixed charge-to-mass ratio, such as DNA or RNA, will separate based on size.
  • the high surface to volume ratio of these capillaries allows application of very high electric fields across the capillary without substantial thermal variation, consequently allowing very rapid separations.
  • these methods provide sensitivity in the range of attomoles, comparable to the sensitivity of radioactive sequencing methods.
  • microcapillary electrophoresis in size separation of nucleic acids has been reported in Woolley and Mathies (Proc. Natl. Acad. Sci. USA (1994) 91 : 1 1348-1 1352), which is incorporated by reference.
  • Capillaries are optionally fabricated from fused silica, or etched, machined, or molded into planar substrates.
  • the capillaries are filled with an appropriate separation/sieving matrix.
  • sieving matrices are known in the art that may be used for this application, including, e.g., hydroxyethyl cellulose, polyacryl amide, agarose, and the like.
  • the specific gel matrix, running buffers and running conditions are selected to obtain the separation required for a particular application.
  • Factors that are considered include, e.g., sizes of the nucleic acid fragments, level of resolution, or the presence of undenatured nucleic acid molecules.
  • running buffers may include agents such as urea to denature double-stranded nucleic acids in a sample.
  • Microfluidic systems for separating molecules such as DNA and RNA are commercially available and are optionally employed in the methods of the present invention.
  • the "Personal Laboratory System” and the “High Throughput System” have been developed by Caliper Lifesciences Corp. (Mountain View, CA).
  • the Agilent 2100 which uses Caliper Lifesciences' LabChipTM microfluidic systems, is available from Agilent Technologies (Palo Alto, CA, USA).
  • Currently, specialized microfluidic devices, which provide for rapid separation and analysis of both DNA and RNA are available from Caliper Lifesciences for the Agilent 2100.
  • chromatographic techniques may be employed for resolving amplification products.
  • Many types of physical or chemical characteristics may be used to effect chromatographic separation in the present invention, including adsorption, partitioning (such as reverse phase), ion-exchange, and size exclusion.
  • cDNA products are captured by their affinity for certain substrates, or other incorporated binding properties.
  • labeled cDNA products such as biotin or antigen can be captured with beads bearing avidin or antibody, respectively.
  • Affinity capture is utilized on a solid support to enable physical separation.
  • solid supports are known in the art that would be applicable to the present invention. Examples include beads (e.g. solid, porous, magnetic), surfaces (e.g.
  • Certain separation embodiments entail the use of microfluidic techniques. Technologies include separation on a microcapillary platform, such as designed by ACLARA BioSciences Inc. (Mountain View, CA), or the LabChipTM microfluidic devices made by Caliper Lifesciences Corp. Another technology developed by ACLARA BioSciences Inc. (Mountain View, CA), or the LabChipTM microfluidic devices made by Caliper Lifesciences Corp. Another technology developed by ACLARA BioSciences Inc. (Mountain View, CA), or the LabChipTM microfluidic devices made by Caliper Lifesciences Corp. Another technology developed by ACLARA BioSciences Inc. (Mountain View, CA), or the LabChipTM microfluidic devices made by Caliper Lifesciences Corp. Another technology developed by ACLARA BioSciences Inc. (Mountain View, CA), or the LabChipTM microfluidic devices made by Caliper Lifesciences Corp. Another technology developed by ACLARA BioSciences Inc. (Mounta
  • Nanogen, Inc. utilizes microelectronics to move and concentrate biological molecules on a semiconductor microchip.
  • Primers are useful both as reagents for hybridization in solution, such as priming PCR amplification, as well as for embodiments employing a solid phase, such as microarrays.
  • sample nucleic acids such as mRNA or DNA are fixed on a selected matrix or surface.
  • PCR products may be attached to the solid surface via one of the amplification primers, then denatured to provide single- stranded DNA.
  • This spatially-partitioned, single-stranded nucleic acid is then subject to hybridization with selected probes under conditions that allow a quantitative determination of target abundance.
  • amplification products from each individual reaction are not physically separated, but are differentiated by hybridizing with a set of probes that are differentially labeled.
  • unextended amplification primers may be physically immobilized at discreet positions on the solid support, then hybridized with the products of a nucleic acid amplification for quantitation of distinct species within the sample.
  • amplification products are separated by way of hybridization with probes that are spatially separated on the solid support.
  • Separation platforms may optionally be coupled to utilize two different separation methodologies, thereby increasing the multiplexing capacity of reactions beyond that which can be obtained by separation in a single dimension.
  • some of the RT-PCR primers of a multiplex reaction may be coupled with a moiety that allows affinity capture, while other primers remain unmodified.
  • Samples are then passed through an affinity chromatography column to separate PCR products arising from these two classes of primers. Flow-through fractions are collected and the bound fraction eluted. Each fraction may then be further separated based on other criteria, such as size, to identify individual components. Detection Methods
  • one or more of the amplicons are detected and/or quantitated.
  • Some embodiments of the methods of the present invention enable direct detection of products.
  • Other embodiments detect reaction products via a label associated with one or more of the amplification primers.
  • labels suitable for use in the present invention are known in the art, including chemiluminescent, isotopic, fluorescent, electrochemical, inferred, or mass labels, or enzyme tags.
  • separation and detection may be a multi-step process in which samples are fractionated according to more than one property of the products, and detected one or more stages during the separation process.
  • An exemplary embodiment of the invention that does not use labeling or modification of the molecules being analyzed is detection of the mass-to-charge ratio of the molecule itself. This detection technique is optionally used when the separation platform is a mass spectrometer.
  • An embodiment for increasing resolution and throughput with mass detection is in mass-modifying the amplification products. Nucleic acids can be mass-modified through either the amplification primer or the chain-elongating nucleoside triphosphates. Alternatively, the product mass can be shifted without modification of the individual nucleic acid components, by instead varying the number of bases in the primers.
  • moieties have been shown to be compatible with analysis by mass spectrometry, including polyethylene glycol, halogens, alkyl, aryl, or aralkyl moieties, peptides (described in, for example, U.S. Patent No. 5,691,141, which is incorporated by reference).
  • Isotopic variants of specified atoms such as radioisotopes or stable, higher mass isotopes, are also used to vary the mass of the amplification product. Radioisotopes can be detected based on the energy released when they decay, and numerous applications of their use are generally known in the art.
  • Stable (non-decaying) heavy isotopes can be detected based on the resulting shift in mass, and are useful for distinguishing between two amplification products that would otherwise have similar or equal masses.
  • Other embodiments of detection that make use of inherent properties of the molecule being analyzed include ultraviolet light absorption (UV) or electrochemical detection. Electrochemical detection is based on oxidation or reduction of a chemical compound to which a voltage has been applied. Electrons are either donated (oxidation) or accepted (reduction), which can be monitored as current. For both UV absorption and electrochemical detection, sensitivity for each individual nucleotide varies depending on the component base, but with molecules of sufficient length this bias is insignificant, and detection levels can be taken as a direct reflection of overall nucleic acid content.
  • Some embodiments of the invention include identifying molecules indirectly by detection of an associated label.
  • a number of labels may be employed that provide a fluorescent signal for detection. If a sufficient quantity of a given species is generated in a reaction, and the mode of detection has sufficient sensitivity, then some fluorescent molecules may be incorporated into one or more of the primers used for amplification, generating a signal strength proportional to the concentration of DNA molecules.
  • fluorescent moieties including Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, carboxyfluorescein, Cascade
  • ET dyes The signal strength obtained from fluorescent dyes can be enhanced through use of related compounds called energy transfer (ET) fluorescent dyes.
  • ET dyes After absorbing light, ET dyes have emission spectra that allow them to serve as "donors" to a secondary "acceptor” dye that will absorb the emitted light and emit a lower energy fluorescent signal.
  • ET dyes include the ABI PRISM BigDye terminators, recently commercialized by Perkin-Elmer Corporation (Foster City,
  • chromophores incorporate the donor and acceptor dyes into a single molecule and an energy transfer linker couples a donor fluorescein to a dichlororhodamine acceptor dye, and the complex is attached, e.g., to a primer.
  • Fluorescent signals can also be generated by non-covalent intercalation of fluorescent dyes into nucleic acids after their synthesis and prior to separation. This type of signal will vary in intensity as a function of the length of the species being detected, and thus signal intensities must be normalized based on size.
  • Several applicable dyes are known in the art, including, but not limited to, ethidium bromide and Vistra Green.
  • intercalating dyes such as YOYO or TOTO
  • YOYO or TOTO Some intercalating dyes, such as YOYO or TOTO, bind so strongly that separate DNA molecules can each be bound with a different dye and then pooled, and the dyes will not exchange between DNA species. This enables mixing separately generated reactions in order to increase multiplexing during analysis.
  • technologies such as the use of nanocrystals as a fluorescent DNA label (Alivisatos, et al. (1996) Nature 382:609-11, which is incorporated by reference) can be employed in the methods of the present invention.
  • Another method described by Mazumder, et al. (Nucleic Acids Res.
  • both electrochemical and infrared methods of detection can be amplified over the levels inherent to nucleic acid molecules through attachment of EC or IR labels.
  • Their characteristics and use as labels are described in, for example, PCT publication WO 97/27327, which is incorporated by reference.
  • Enzyme-linked reactions are also employed in the detecting step of the methods of the present invention. Enzyme-linked reactions theoretically yield an infinite signal, due to amplification of the signal by enzymatic activity.
  • an enzyme is linked to a secondary group that has a strong binding affinity to the molecule of interest. Following separation of the nucleic acid products, enzyme is bound via this affinity interaction. Nucleic acids are then detected by a chemical reaction catalyzed by the associated enzyme.
  • a primer may be synthesized containing a biotin molecule. After amplification, amplicons are separated by size, and those made with the biotinylated primer are detected by binding with streptavidin that is covalently coupled to an enzyme, such as alkaline phosphatase. A subsequent chemical reaction is conducted, detecting bound enzyme by monitoring the reaction product.
  • the secondary affinity group may also be coupled to an enzymatic substrate, which is detected by incubation with unbound enzyme.
  • Exploitation of known high-affinity biological interactions can provide a mechanism for physical capture.
  • Some examples of high-affinity interactions include those between a hormone with its receptor, a sugar with a lectin, avidin and biotin, or an antigen with its antibody.
  • affinity capture molecules are retrieved by cleavage, denaturation, or eluting with a competitor for binding, and then detected as usual by monitoring an associated label.
  • the binding interaction providing for capture may also serve as the mechanism of detection.
  • an amplification product or products are optionally changed, or "shifted,” in order to better resolve the amplification products from other products prior to detection.
  • chemically cleavable primers can be used in the amplification reaction.
  • one or more of the primers used in amplification contains a chemical linkage that can be broken, generating two separate fragments from the primer. Cleavage is performed after the amplification reaction, removing a fixed number of nucleotides from the 5' end of products made from that primer. Design and use of such primers is described in detail in, for example, PCT publication WO 96/37630, which is incorporated by reference.
  • the statistical significance of markers as expressed in q orp values based on the concept of the false discovery rate is optionally determined. In doing so, a measure of statistical significance called the q value is associated with each tested feature.
  • the q value is similar to thep value, except it is a measure of significance in terms of the false discovery rate rather than the false positive rate (see, e.g., Storey et al. (2003) Proc.Natl.Acad.Sci. 100:9440- 5, which is incorporated by reference).
  • markers may have ⁇ -values of less than about 3E-06, typically less than about 1.5E-09, more typically less than about 1.5E-1 1, even more typically less than about 1.5E-20, and still more typically less than about 1.5E-30.
  • the expression level of at least about two, typically of at least about ten, more typically of at least about 25, and even more typically of at least about 50 markers is determined as described herein or by another technique known to those of skill in the art.
  • expression levels of each of these genes in a sample is determined and compared with expression levels detected in one or more reference or control cells. For example, the International Publication No.
  • WO 03/039443 discloses certain marker genes the expression levels of which are characteristic for certain leukemias. Certain of the markers and/or methods disclosed therein are optionally utilized in performing the methods described herein.
  • the level of the expression of a marker may be indicative of the disease, genetic disorder, or other condition of a cell or tissue under consideration.
  • the level of expression of a marker or group of markers is measured and is generally compared with the level of expression of the same marker or the same group of markers from other cells or samples. The comparison may be effected in an actual experiment or in silico. There is a meaningful difference in these levels of expression, e.g., when these expression levels (also referred to as expression pattern, expression signature, or expression profile) are measurably different.
  • the difference is typically at least about 5%, 10% or 20%, more typically at least about 50% or may even be as high as 75% or 100%.
  • the difference in the level of expression is optionally at least about 200%, i.e., two fold, at least about 500%, i.e., five fold, or at least about 1000%, i.e., 10 fold in some embodiments.
  • the expression level of markers expressed lower in a first subtype than in at least one second subtype, which differs from the first subtype is at least about 5%, 10% or 20%, more typically at least about 50% or may even be about 75% or about 100%, more typically at least about 10-fold, even more typically at least 50-fold, and still more typically at least about 100-fold lower in the first subtype.
  • the expression level of markers expressed higher in a first subtype than in at least one second subtype, which differs from the first subtype is at generally least about 5%, 10% or 20%, more generally at least about 50% or may even be about 75% or about 100%, more generally at least 10-fold, still more generally at least about 50-fold, and even more generally at least about 100-fold higher in the first subtype.
  • the classification accuracy of a given gene list for a set of microarray experiments is preferably estimated using Support Vector Machines (SVM), because there is evidence that SVM-based prediction slightly outperforms other classification techniques, such as k-Nearest Neighbors (k-NN).
  • SVM Support Vector Machines
  • the LIBSVM software package version 2.36 for example, is optionally used (SVM-type: SVC, linear kernel (http://www.csie.ntu.edu.tw/-cj lin/libsvrn/)).
  • Machine learning algorithms are also described in, e.g., Brown et al. (2000) Proc.Natl.Acad.Sci.. 97:262-267, Furey et al. (2000) Bioinformatics. 16 : 906-914, and Vapnik, Statistical Learning Theory,
  • the classification accuracy of a given gene list for a set of microarray experiments can be estimated using Support Vector Machines (SVM) as supervised learning techniques.
  • SVMs are trained using differentially expressed genes, which were identified on a subset of the data and then this trained model is employed to assign new samples to those trained groups from a second and different data set.
  • Differentially expressed genes are optionally identified, e.g., applying analysis of variance (ANOVA) and t-test-statistics (Welch t-test).
  • ANOVA analysis of variance
  • Weight t-test t-test-statistics
  • respective training sets consisting of, e.g., 2/3 of cases and test sets with 1/3 of cases to assess classification accuracies can be designated. Assignment of cases to training and test sets is optionally randomized and balanced by diagnosis.
  • SVM Support Vector Machine
  • the apparent accuracy of prediction i.e., the overall rate of correct predictions of the complete data set can be estimated by, e.g., 10-fold cross validation.
  • This process typically includes dividing the data set into 10 approximately equally sized subsets, training an SVM-model for 9 subsets, and generating predictions for the remaining subset. This training and prediction process can be repeated 10 times to include predictions for each subset. Subsequently the data set can be split into a training set, consisting of two thirds of the samples, and a test set with the remaining one third. Apparent accuracy for the training set can also be estimated by 10-fold cross validation (analogous to apparent accuracy for complete set).
  • An SVM-model of the training set is optionally built to predict diagnosis in the independent test set, thereby estimating true accuracy of the prediction model.
  • Sensitivity (number of positive samples predicted)/(number of true positive)
  • Specificity (number of negative samples predicted)/(number of true negatives).
  • Gene expression profiling is a promising technology with respect to future applications in routine clinical procedures. This is true for initial diagnosis of a disease and for the determination of a patient's prognosis. 1'10 As a pioneering application in genomic medicine, the diagnosis of leukemias has been proposed.”
  • Important examples are a molecularly designed BCR-ABL kinase inhibitor (Imatinib mesylate) for the treatment of t(9;22)(q34;ql 1) positive chronic myeloid leukemia (CML) and all-trans retinoic acid (ATRA) for the treatment of acute promyelocyte leukemia (APL) with translocation t(15;17)(q22;ql2).
  • Imatinib mesylate for the treatment of t(9;22)(q34;ql 1
  • CML chronic myeloid leukemia
  • ATRA all-trans retinoic acid
  • APL acute promyelocyte leukemia
  • AML acute myeloid leukemia
  • microarray studies clearly correlated specific gene expression signatures to the four recurrent chromosomal aberrations t(15;17), t(8;21), inv(16), and t(l Iq23)/MLL.
  • Both approaches using minimal sets of genes from supervised methods and unsupervised algorithms accurately identified gene signatures associated with AML and recurrent chromosomal aberrations.
  • ALL acute lymphoblastic leukemia
  • the robustness of diagnostic gene expression patterns was approached in this analysis by visualizing gene signatures according to various parameters.
  • the samples were given by ordering according to: (A) Duration of sample shipment; (B) 375' ratio of GAPD hybridization signals as a measure of RNA quality; (C) Duration of sample storage time until microarray target preparation; (D) Date of sample target preparation within the marker discovery study; (E) Age of the leukemia patient at diagnosis; and (F) Type of specimen, i.e., bone marrow or peripheral blood.
  • Gene expression profiling is used for the classification of malignant diseases and to identify novel prognostic markers and potential therapeutic targets in a research setting. This analysis further addressed the issue of whether the microarray platform also qualifies for a diagnostic setting which requires robustness of the technique itself and the markers used.
  • microarray platforms e.g. short DNA-oligonucleotide and cDNA microarrays. Platforms have evolved by, e.g., increasing the information content on each array, mainly by shrinking the feature size of represented probes. These differences can cause difficulties in data comparisons and might impair the transferability of, e.g., results from research studies to large-scale clinical trials. Accordingly, this validation study was designed to assess the robustness and accuracy of diagnostic marker patterns in a prospective cohort of patients.
  • a scanner with removable 48-array autoloader carousel maximized array throughput was utilized in the analysis.
  • both array designs were normalized using the recommended set of 100 housekeeping genes (scaling to common target intensity).
  • the Ul 33 set reference data matrix was applied to train the classifier (Support Vector Machines).
  • the 400 independent blinded validation samples were predicted using the set of differentially expressed genes from the training data set.
  • the methods section contains both information on statistical analyses used for identification of differentially expressed genes and detailed annotation data of identified microarray probe sets.
  • sequence data are omitted due to their large size, and because they do not change, whereas the annotation data are updated periodically, for example new information on chromosomal location and functional annotation of the respective gene products. Sequence data are available to download in the NetAffx Download Center on the world wide web at affymetrix.com.
  • Microarray probe sets for example, found to be differentially expressed between different types of leukemia samples are further described by additional information.
  • the fields are of the following types:
  • HG-LJl 33 ProbeSet_ID describes the probe set identifier. Examples are: 200007 _at, 20001 l_s_at,200012_x_at. Sequence Type The Sequence Type indicates whether the sequence is an Exemplar, Consensus or
  • Control sequence An Exemplar is a single nucleotide sequence taken directly from a public database. This sequence could be an mRNA or an expressed sequence tag (EST).
  • EST expressed sequence tag
  • a Consensus sequence is a nucleotide sequence assembled by
  • Affymetrix based on one or more sequence taken from a public database.
  • Transcript ID The cluster identification number with a sub-cluster identifier appended.
  • Sequence Derived From The accession number of the single sequence, or representative sequence on which the probe set is based. Refer to the "Sequence Source” field to determine the database used.
  • Sequence ID For Exemplar sequences: Public accession number or GenBank identifier.
  • Consensus sequences Affymetrix identification number or public accession number.
  • Sequence Source The database from which the sequence used to design this probe set was taken.
  • GenBank® GenBank®, RefSeq, UniGene, TIGR (annotations from The Institute for Genomic Research).
  • Gene Symbol and Title A gene symbol and a short title, when one is available. Such symbols are assigned by different organizations for different species. Affymetrix annotational data comes from the UniGene record. There is no indication which species-specific databank was used, but some of the possibilities include for example HUGO: The Human Genome Organization.
  • Unigene Accession UniGene accession number and cluster type. Cluster type can be "full length" or
  • LocusLink This information represents the LocusLink accession number.
  • EXAMPLE 4 SAMPLE PREPARATION. PROCESSING AND DATA ANALYSIS
  • Microarray analyses were performed utilizing the GeneChip® System (Affymetrix,
  • Hybridization target preparations were performed according to recommended protocols (Affymetrix Technical Manual). More specifically, at time of diagnosis, mononuclear cells were purified by Ficoll-Hypaque density centrifugation. They had been lysed immediately in RLT buffer (Qiagen, Hilden,
  • RNA samples were thawed, homogenized (QIAshredder, Qiagen), and total RNA was extracted (RNeasy Mini Kit, Qiagen). Subsequently, 5-10 ⁇ g total RNA isolated from 1 x 10 7 cells was used as starting material for cDNA synthesis with oligo[(dT) 24 T7promotor] 65 primer (cDNA Synthesis System, Roche Applied Science, Mannheim, Germany). cDNA products were purified by phenol/chloroform/IAA extraction (Ambion, Austin, TX, USA) and acetate/ethanol-precipitated overnight. For detection of the hybridized target nucleic acid biotin-labeled ribonucleotides were incorporated during the following in vitro transcription reaction (Enzo BioArray High Yield
  • RNA Transcript Labeling Kit Enzo Diagnostics
  • 15 ⁇ g cRNA was fragmented by alkaline treatment (200 mM Tris-acetate, pH 8.2/500 mM potassium acetate/150 mM magnesium acetate) and added to the hybridization cocktail sufficient for five hybridizations on standard GeneChip® microarrays (300 ⁇ L final volume). Washing and staining of the probe arrays was performed according to the recommended Fluidics Station protocol (EukGE-WS2v4).
  • Affymetrix Microarray Suite software (version 5.0.1) extracted fluorescence signal intensities from each feature on the microarrays as detected by confocal laser scanning according to the manufacturer's recommendations.
  • Expression analysis quality assessment parameters included visual array inspection of the scanned image for the presence of image artifacts and correct grid alignment for the identification of distinct probe cells as well as both low 375' ratio of housekeeping controls (mean: 1.90 for GAPD) and high percentage of detection calls (mean: 46.3% present called genes).
  • the 3' to 5' ratio of GAPD probesets can be used to assess RNA sample and assay quality. Signal values of the 3' probe sets for GAPD are compared to the Signal values of the corresponding 5' probe set.
  • the ratio of the 3' probe set to the 5' probe set is generally no more than 3.0.
  • a high 3' to 5' ratio may indicate degraded RNA or inefficient synthesis of ds cDNA or biotinylated cRNA (GeneChip Expression Analysis Technical Manual, available on the world wide web at affymetrix.com as of 1 1/4/2004).
  • Detection calls are used to determine whether the transcript of a gene is detected (present) or undetected (absent) and were calculated using default parameters of the Microarray Analysis Suite MAS 5.0 software package.
  • Staudt LM Molecular diagnosis of the hematologic cancers. N.Engl.J.Med. 2003;348:1777-1785. 7. VaIk PJ, Verhaak RG, Beijen MA et al. Prognostically useful gene-expression profiles in acute myeloid leukemia. N.Engl.J.Med. 2004;350:1617-1628.
  • ALL acute lymphoblastic leukemia
  • Kern W Kohlmann A, Wuchter C et al. Correlation of protein expression and gene expression in acute leukemia. Cytometry. 2003;55B:29-36. 18. Kohlmann A, Schoch C, Thomasger S et al. Molecular characterization of acute leukemias by use of microarray technology. Genes Chromosomes. Cancer. 2003;37:396-405.

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L'invention concerne des processus rapides et fiables permettant de valider ou d'évaluer la robustesse des tests d'expression génique.
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