METHODS OF VALIDATING GENE EXPRESSION ASSAYS
FIELD OF THE INVENTION
The present invention relates to gene expression analyses and to methods of validating such analyses.
BACKGROUND OF THE INVENTION 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. In 1976, for example, 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. In addition, genetic abnormalities occurring in leukemic blasts were recognized as having a major impact on the morphological picture and on prognosis. As a consequence, 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. To illustrate, the analysis of the morphology and cytochemistry of bone marrow blasts and peripheral blood cells is commonly used to establish a diagnosis. In some cases, for example, immunophenotyping is also utilized to separate an undifferentiated AML from acute lymphoblastic leukemia and from CLL. In certain instances, leukemia subtypes can be diagnosed by cytomorphology alone, but this typically requires that an expert review sample smears. However, 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. An aim of these techniques, aside from diagnosis, is to determine the prognosis of the leukemia under consideration. One disadvantage of these methods, however, 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. Furthermore, great experience in preparing chromosomes and analyzing karyotypes is generally needed to obtain correct results in most cases. Using these techniques in combination, 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. For example, 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)). In patients treated with this new drug, the therapy response is dramatically higher as compared to other drugs that have previously been used. Another example is a subtype of acute myeloid leukemia, AML M3 and its variant M3v, which both include the karyotype t(15;17)(q22;qll- 12). The introduction of all-trans retinoic acid (ATRA) has improved the outcome in this subgroup of patient from about 50% to 85% long-term survivors. Accordingly, the rapid and accurate identification of distinct leukemia subtypes is of consequence to further drug development in addition to diagnostics and prognostics. According to 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. However, for the analysis of acute leukemias the selection of the two different subgroups was performed using exclusively morphologic-phenotypical criteria. This was only descriptive and did not provide deeper insights into the pathogenesis or the underlying biology of the leukemia. The approach reproduces only very basic knowledge of cytomorphology and intends to differentiate classes. However, the data generated via such an approach is generally not sufficient to predict prognostically relevant cytogenetic aberrations.
SUMMARY OF THE INVENTION The present invention relates to rapid, cost effective, and reliable approaches to validating or assessing the robustness of a given gene expression analysis. In one aspect, for example, 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. In some embodiments, 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. In addition, 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. In some embodiments, 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. In certain embodiments, 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.
Essentially 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. To illustrate, the measure of RNA quality optionally comprises a 375' ratio of GAPD hybridization signals. To further illustrate, 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. In some embodiments, for example, the expression level is detected using an array, a robotics system, and/or a microfluidic device. In certain embodiments, 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. In these embodiments, 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. In certain embodiments, 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.
BRIEF DESCRIPTION OF THE DRAWINGS Figures IA-F. Pattern robustness in AML with recurrent chromosomal aberrations. Hierarchical cluster analysis of adult AML samples with recurrent chromosomal aberrations based on Affymetrix U 133 chip design microarray expression data. This analysis was based on a subset of n=287 probe sets which were identified to be differentially expressed when analyzing AML samples with t(15;17), t(8;21), inv(16), and t(l Iq23)/MLL. In the pattern the normalized expression value for each gene is coded by color (standard deviation from mean). Red cells indicate high expression and green cells indicate low expression. Only the genes (rows) were clustered by the algorithm. For each AML subtype the samples (columns) are given by ordering according to various parameters. (Figure IA) Duration of sample shipment (hours, days). (Figure IB) 375' ratio of GAPD hybridization signals (range; median). (Figure 1C) Duration of sample storage until microarray target preparation (range; months). (Figure ID) Date of sample target preparation within marker discovery study (range; months). (Figure IE) Type of specimen. (Figure IF) Age of the patient at diagnosis.
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. (Figure 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.
DETAILED DESCRIPTION
DEFINITIONS
Before describing the present invention in detail, it is to be understood that this invention is not limited to particular embodiments. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Units, prefixes, and symbols are denoted in the forms suggested by the International System of Units (SI), unless specified otherwise. Numeric ranges are inclusive of the numbers defining the range. As used in this specification and the appended claims, the singular forms "a", "an" and "the" also include plural referents unless the context clearly dictates otherwise. To illustrate, reference to "a cell" includes two or more cells. Further, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The terms defined below, and grammatical variants thereof, are more fully defined by reference to the specification in its entirety.
"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.
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. (1982) Nature 295:712); Fab proteins including Fab' and F(ab')2 fragments whether covalently or non-covalently aggregated; light or heavy chains alone, typically variable heavy and light chain regions (VH and VL regions), and more typically including the hypervariable regions (otherwise known as the complementarity determining regions (CDRs) of the VH and VL regions); F0 proteins; "hybrid" antibodies capable of binding more than one antigen; constant-variable region chimeras; "composite" immunoglobulins with heavy and light chains of different origins; "altered" antibodies with improved specificity and other characteristics as prepared by standard recombinant techniques, by mutagenic techniques, or other directed evolutionary techniques known in the art. Derivatives of antibodies include scFvs, chimeric and humanized antibodies. See, e.g., Harlow and Lane, Antibodies, a laboratory manual. CSH Press (1988), which is incorporated by reference. For the detection of polypeptides using antibodies or fragments thereof, there are a variety of 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). To facilitate detection, an antibody is typically labeled by one or more of the labels described herein or otherwise known to persons skilled in the art.
In general, 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. The person skilled in the art knows a collection of nucleic acids or polypeptide spotted onto a substrate/solid support also under the term "array". As also known to the person skilled in the art, 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 cm2. Furthermore, where appropriate 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. Generally, 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. Or, 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. According to the present invention, 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. For carrying out the methods of present invention 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.
The term "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. Such differences may be evidenced by a change in mRNA levels, surface expression, secretion or other partitioning of a polypeptide, for example. 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. In certain embodiments, "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.
The term "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. The term
"determining the expression level" refers to the determination of the level of expression of one or more markers.
The term "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.
The term "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.).
The term "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.
Nucleic acids "hybridize" when they associate, typically in solution. Nucleic acids hybridize due to a variety of well-characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. In certain embodiments, hybridization occurs under conventional hybridization conditions, such as under stringent conditions as described, for example, in Sambrook et al., in "Molecular Cloning: A Laboratory Manual" (1989), Eds. J. Sambrook, E. F. Fritsch and T. Maniatis, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, which is incorporated by reference. Such conditions are, for example, hybridization in 6x SSC, pH 7.0 / 0.1 % SDS at about 450C for 18-23 hours, followed by a washing step with 2x SSC/1 % SDS at 50°C. In order to select the stringency, 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 500C for high stringency. In addition, the temperature of the washing step can be varied between room temperature (ca. 220C), for low stringency, and 650C to 700C for high stringency. Also contemplated are 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. For example, lower stringency conditions include an overnight incubation at 37°C in a solution comprising 6X SSPE (2OX SSPE = 3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 mg/mL salmon sperm blocking DNA, followed by washes at 500C with 1 X SSPE, 0.1%
SDS. In addition, to achieve even lower stringency, 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. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology- Hybridization with Nucleic Acid Probes part I chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," (Elsevier, New York), as well as in Ausubel (Ed.) Current Protocols in Molecular Biology, Volumes I, II, and III, (1997), which are each incorporated by reference. Hames and Higgins (1995) Gene Probes 1 IRL Press at Oxford University Press, Oxford, England, (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probes 2 IRL Press at Oxford University Press, Oxford, England (Hames and Higgins 2) provide details on the synthesis, labeling, detection and quantification of DNA and RNA, including oligonucleotides. Both Hames and Higgins 1 and 2 are incorporated by reference.
"inv(16)" refers to AML with inversion 16 according to the WHO classification of haematological malignancies.
A "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.). Exemplary labels include fluorescent labels (including, e.g., quenchers or absorbers), non- fluorescent labels, colorimetric labels, chemiluminescent labels, bioluminescent labels, radioactive labels (such as3H,35S,32P,1251,57Co or14C), mass-modifying groups, antibodies, antigens, biotin, haptens, digoxigenin, enzymes (including, e.g., peroxidase, phosphatase, etc.), and the like. To further illustrate, 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. (Hatfield, PA, USA)). In some embodiments, 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. Similarly, 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. In some embodiments, "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.
The term "marker" 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. In certain embodiments, for example, "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. In particular, 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). The 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. In some embodiments, the expression level of a marker is determined by the deteπnining the expression of its corresponding polynucleotide.
The term "normal karyotype" refers to a state of those cells lacking any visible karyotype abnormality detectable with chromosome banding analysis.
The term "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 (LNA™s), and the like. In certain applications, 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.) 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. For example, 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). Similarly, and when desirable, the sequences can be labeled using methodologies well known in the art such as described in U.S. patent application numbers 5,464,746;
5,424,414; and 4,948,882 all of which are incorporated by reference.
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. For example, a polynucleotide of the invention can contain one or more modified, non-standard, or derivatized base moieties, including, but not limited to, N6-methyl-adenine, N6-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- methylcytosine, N6-methyl adenine, 7-methylguanine, 5-methylaminomethyluracil,
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. Other examples of modified, non-standard, or dervatized base moieties may be found in U.S. Patent Nos. 6,001 ,611, 5,955,589, 5,844,106, 5,789,562, 5,750,343, 5,728,525, and 5,679,785, each of which is incorporated by reference. Furthermore, a 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.
The term "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. In some embodiments, polynucleotides also include any fragment (or complementary sequence thereto) of a sequence corresponding to or derived from any of the markers defined herein.
The term "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. The term "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
Northern blot, and the like. In certain embodiments, 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. For example, 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. 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 (e.g., primers, probes, etc.), whether hybridization assay probes, amplification primers, or helper oligonucleotides, may be modified with chemical groups to enhance their performance or to facilitate the characterization of amplification products. For example, 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",
EP 0 313 219, which is incorporated by reference) incorporated between nucleotides in the nucleic acid chain which do not interfere with hybridization or the elongation of the primer. 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. A "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. In some embodiments, 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. For example, 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. Typically, the organism is a mammalian organism, particularly a human organism.
The term "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.
The term "target" refers to an object that is the subject of analysis. In some embodiments, for example, targets are specific nucleic acid sequences (e.g., mRNAs of expressed genes, etc.), the presence, absence or abundance of which are to be determined. In certain embodiments, targets include polypeptides (e.g., proteins, etc.) of expressed genes. Typically, the sequences subjected to analysis are in or derived from "target cells", such as a particular type of leukemia cell.
INTRODUCTION Before a new technology can be used in, e.g., a diagnostic setting it generally has to be shown that the methodology can provide robust results. With respect to microarrays, for example, various parameters such as 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. In some embodiments, for example, 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). 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. In some embodiments, 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. In certain embodiments, 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. In other embodiments, for example, 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. These techniques are well known and are explained in, for example, Current Protocols in Molecular Biology, Volumes I, II, and III, 1997 (F. M. Ausubel ed.); Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001 ; Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc.,
San Diego, CA (Berger), DNA Cloning: A Practical Approach. Volumes I and II, 1985 (D. N. Glover ed.); Oligonucleotide Synthesis. 1984 (M. L. Gait ed.); Nucleic Acid Hybridization, 1985, (Hames and Higgins); Transcription and Translation, 1984 (Hames and Higgins eds.); Animal Cell Culture, 1986 (Freshney ed.); Immobilized Cells and Enzymes, 1986 (IRL Press); Perbal, 1984, A Practical
Guide to Molecular Cloning; the series, Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells, 1987 (J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory); Greer et al. (Eds.), Wintrobe's Clinical Hematology, 11th Ed., Lippincott Williams & Wilkins (2003); Shirlyn et al., Clinical Laboratory Hematology, Prentice Hall (2002); Lichtman et al., Williams
Manual of Hematology, 6th Ed., McGraw-Hill Professional (2002); and Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively), all of which are incorporated by reference.
SAMPLECOLLECTIONANDPREPARATION Samples are collected and prepared for analysis using essentially any technique known to those of skill in the art. In addition, samples are optionally obtained from essentially any source. In certain embodiments, for example, blood samples are obtained from subjects via venipuncture. Whole blood specimens are optionally collected in EDTA, Heparin or ACD vacutainer tubes. In other embodiments, 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., -800C 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 &
Wilkins (1998), and Slockbower et al. (Eds.), Collection and Handling of Laboratory Specimens: A Practical Guide, Lippincott Williams & Wilkins (1983), which are both incorporated by reference.
Treatment of Cells
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. For example, 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. Optionally, 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. 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).
RNA Isolation
In some embodiments, 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. In certain embodiments, 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. Optionally, 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)). Alternatively,
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.
Alternatively, 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. When using crude cellular lysates, genomic DNA could contribute one or more copies of target sequence, depending on the sample. In situations in which the target sequence is derived from one or more highly expressed genes, 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.
One skilled in the art can design a variety of specialized priming applications that would facilitate use of crude extracts as samples for the purposes of this invention.
GENE EXPRESSION PROFILING
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.
Essentially 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. To illustrate, commonly used methods for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization
(Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)), RNAse protection assays (Hod, Biotechniques 13:852-854 (1992)), and reverse transcription polymerase chain reaction (RT-PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)). Alternatively, 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). Optionally, molecular species, such as antibodies, aptamers, etc. that can specifically bind to proteins or fragments thereof are used for analysis (see, e.g., Beilharz et al., Brief Funct Genomic Proteomic 3(2):103-l 11 (2004)). Some of these techniques, with a certain degree of overlap in some cases, are described further below.
In certain embodiments, for example, the methods described herein include determining the expression levels of transcribed polynucleotides. In some of these embodiments, 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. In order to determine the expression level of transcribed polynucleotides, 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.
In some embodiments, 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.
No. 5,210,015, U.S. Pat. No. 5,804,375, and U.S. Pat. No. 5,487,972, which are each incorporated by reference. In some embodiments, for example, 5 '-nuclease methods utilize the exonuclease activity of certain polymerases to generate signals. In these approaches, 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. Other 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.
In one protocol, the marker, i.e., the polynucleotide, 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.
5,945,334, EP 0 619 321 , and EP 0 373 203, which are each incorporated by reference.
In another embodiment, 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.
These and other exemplary gene expression profiling techniques are described further below.
Blotting Techniques
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 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)).
Assays Based on Protection from Enzymatic Degradation Techniques to monitor RNA that make use of protection from enzymatic degradation include Sl analysis and RNAse protection assays (RPAs). Both of these 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.
Reverse Transcriptase PCR (RT-PCR) and Real-Time Detection
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. These 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.
General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., supra. Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987), and De Andres et al.,
BioTechniques 18:42044 (1995), which are each incorporated by reference. In particular, RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions. For example, total RNA from cells in culture can be isolated using Qiagen Rneasy® mini-columns (referred to above). Other commercially available RNA isolation kits include MasterPure™ Complete DNA and RNA Purification Kit (EPICENTRE™, 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.
Since 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). 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. For example, 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. Although 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. Thus, 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. During the amplification reaction, 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,
Mannheim, Germany) or an ABI PRISM 7700™ Sequence Detection System™ (Perkin-Elmer-Applied Biosystems, Foster City, CA, USA).
To minimize errors and the effect of sample-to-sample variation, 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 β- 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. 5,599,672; Liang and Pardee (1992) Science 257:967-971 ; Welsh et al. (1992) Nucleic Acids Res. 20:4965-4970, which are each incorporated by reference). Both methods use random priming to generate RT-PCR fingerprint profiles of transcripts in an unfractionated RNA preparation. The signal generated in these types of analyses is a pattern of bands separated on a sequencing gel. Differentially expressed genes appear as changes in the fingerprint profiles between two samples, which can be loaded in separate wells of the same gel. This type of readout allows identification of both up- and down-regulation of genes in the same reaction, appearing as either an increase or decrease in intensity of a band from one sample to another.
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. To illustrate, one example 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. When the molecular beacon is present free in solution, i.e., not hybridized to a second nucleic acid, 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. (1995) "Molecular beacon probes combined with amplification by NASBA enable homogenous real-time detection of RNA," Nucleic Acids Res. 26:2150-2155; Kostrikis et al. (1998) "Molecular beacons: spectral genotyping of human alleles"
Science 279:1228-1229; Fang et al. (1999) "Designing a novel molecular beacon for surface-immobilized DNA hybridization studies" J. Am. Chem. Soc. 121 :2921- 2922; and Marras et al. (1999) "Multiplex detection of single-nucleotide variation using molecular beacons" Genet. Anal. Biomol. Eng. 14:151-156, all of which are incorporated by reference. A variety of commercial suppliers produce standard and custom molecular beacons, including Oswel Research Products Ltd. (UK), Research Genetics (a division of Invitrogen, Huntsville, AL, USA), the Midland Certified Reagent Company (Midland, TX, USA), and Gorilla Genomics, LLC (Alameda, CA, USA). A variety of kits which utilize molecular beacons are also commercially available, such as the Sentinel™ Molecular Beacon Allelic
Discrimination Kits from Stratagene (La Jolla, CA, USA) and various kits from Eurogentec SA (Belgium) and Isogen Bioscience BV (Netherlands).
Nucleic Acid Array-Based Analysis
Differential gene expression can also be identified, or confirmed using arrayed oligonucleotides (e.g., microarrays), which have the benefit of assaying for sample hybridization to a large number of probes in a highly parallel fashion. In these approaches, polynucleotide sequences of interest (e.g., probes, such as cDNAs, mRNAs, oligonucleotides, etc.) are plated, synthesized, or otherwise disposed on a microchip substrate or other type of solid support (see, e.g., U.S. Patent Nos. 5,143,854 and 5,807,522; Fodor et al. (1991) Science 251 :767-773; and Schena et al. (1995) Science 270:467-470, which are each incorporated by reference). 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. As in the RT- PCR assays referred to above, the source of mRNA typically is total RNA isolated from a sample.
In certain embodiments, 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. For this purpose, selected 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. 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.
To illustrate other embodiments of microarray-based assays, PCR amplified inserts of cDNA clones are applied to a substrate in a dense array. In some embodiments, for example, 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. With 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. USA 93(2):106-149 (1996), which is incorporated by reference). Other 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. For example, 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. Upon applying appropriate washing steps, those cDNAs that have a counterpart in the oligonucleotide panel or array will be detected (e.g., quantitatively detected). Various advantageous embodiments of this general method are feasible. For example, mRNA or cDNA may be amplified, e.g., by a polymerase chain reaction or another nucleic acid amplification technique. In some embodiments, where quantitative assessments are sought, it is generally desirable that the number of amplified copies corresponds to the number of mRNAs originally present in the cell. Optionally, cDNAs are transcribed into cRNAs prior to hybridization steps in a given assay. In these embodiments, labels can be attached or incorporated cRNAs during or after the transcription step. To further illustrate, one exemplary embodiment of the methods of the invention includes, as follows (1) obtaining a sample, e.g. bone marrow or peripheral blood aliquots, from a patient; (2) extracting RNA, e.g., mRNA, from the sample; (3) reverse transcribing the RNA into cDNA; (4) in vitro transcribing the cDNA into cRNA; (5) fragmenting the cRNA; (6) hybridizing the fragmented cRNA on selected microarrays (e.g., the HG-Ul 33 microarray set available from Affymetrix, Inc. (Santa Clara, CA USA)); and (7) detecting hybridization.
Serical Analysis of Gene Expression (SAGE)
Serial analysis of gene expression (SAGE) 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. Initially, a short sequence tag (e.g., about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript. Then, 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.
Gene Expression Analysis by Massively Parallel Signature
Sequencing (MPSS)
These sequencing approaches generally combine non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 μm diameter microbeads. Typically, 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 106 microbeads/cm2). 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.
Immunoassays and proteomics
Essentially 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. To illustrate, immunohistochemical methods are optionally used for detecting the expression levels of the targets described herein. Thus, antibodies or antisera (e.g., polyclonal antisera) and in certain embodiments, monoclonal antibodies specific for particular targets are used to detect expression. In some of these embodiments, 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. Such 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.
Additional labels are described further herein. Alternatively, 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.
To further illustrate, 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. To further illustrate, 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-Plex™ platform available from BIO-RAD Laboratories (Munich, Germany). In some embodiments of the invention, 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. These 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.
Aptamers can be identified using the SELEX process (Gold (1996) "The SELEX process: a surprising source of therapeutic and diagnostic compounds," Harvey
Lect. 91 :47-57, which is incorporated by reference). Aptamer arrays are commercially available from various suppliers including, e.g., SomaLogic, Inc.
(Boulder, CO, USA).
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,"
Expert Rev. MoI. Diagn. 1(1): 1 1-18, which are each incorporated by reference. Protein chips and related instrumentation are available from commercial suppliers, such as Ciphergen Biosystems, Inc. (Fremont, CA, USA).
OLIGONUCLEOTIDE PREPARATION
Various approaches can be utilized by one of skill in the art to design oligonucleotides for use as probes and/or primers. To illustrate, the DNAstar software package available from DNASTAR, Inc. (Madison, WI) can be used for sequence alignments. For example, 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. To further illustrate, 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
85% identity with one another are typically identified and oligonucleotide sequences in these regions can be selected. Many other sequence alignment algorithms and software packages are also optionally utilized. 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.,
Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press (1998), which are both incorporated by reference.
To further illustrate, 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 &
Wunsch (1970) J. MoI. Biol. 48:443, by the search for similarity method of Pearson & Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, which are each incorporated by reference, and by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (Madison, WI)), or by even by visual inspection. Another example algorithm that is suitable for determining percent sequence identity is the BLAST algorithm, which is described in, e.g., Altschul et al. (1990) J. MoI. Biol. 215:403-410, which is incorporated by reference. Software for performing versions of BLAST analyses is publicly available through the National Center for Biotechnology Information on the world wide web at ncbi.nlm.nih.gov/ as of October 22, 2004.
An additional example of a useful sequence alignment algorithm is PILEUP. 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. In certain embodiments, for example, 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. To further illustrate, oligonucleotides can also be synthesized using a triester method (see, e.g., Capaldi et al. (2000) "Highly efficient solid phase synthesis of oligonucleotide analogs containing phosphorodithioate linkages" Nucleic Acids Res. 28(9):e40 and Eldrup et al. (1994) "Preparation of oligodeoxyribonucleoside phosphorodithioates by a triester method" Nucleic Acids Res. 22(10): 1797-1804, which are both incorporated by reference). Other synthesis techniques known in the art can also be utilized, including, e.g., using an automated synthesizer, as described in
Needham-VanDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168, which is incorporated by reference. A wide variety of equipment is commercially available for automated oligonucleotide synthesis. Multi-nucleotide synthesis approaches (e.g., tri-nucleotide synthesis, etc.) are also optionally utilized. Moreover, the primer nucleic acids optionally include various modifications. In certain embodiments, for example, primers include restriction site linkers, e.g., to facilitate subsequent amplicon cloning or the like. To further illustrate, primers are also optionally modified to improve the specificity of amplification reactions as described in, e.g., U.S. Pat. No. 6,001,611, entitled "MODIFIED NUCLEIC ACID AMPLIFICATION PRIMERS," issued December 14, 1999 to Will, which is incorporated by reference. Primers and probes can also be synthesized with various other modifications as described herein or as otherwise known in the art.
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. In general, 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. Using commercially available phosphoramidite reagents, one can produce oligonucleotides containing functional groups (e.g., thiols or primary amines) at either the 5' or 3' terminus via an appropriately protected phosphoramidite, and 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.
Essentially any labeling moiety is optionally utilized to label a probe and/or primer by techniques well known in the art. In some embodiments, for example, 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., BIGDYE™ v 1 dyes, BIGDYE™ v 2 dyes, BIGDYE™ v 3 dyes, etc.), Lucifer dyes (e.g., Lucifer yellow, etc.), CASCADE
BLUE®, Oregon Green, and the like. Additional examples of fluorescent dyes are provided in, e.g., Haugland, Molecular Probes Handbook of Fluorescent Probes and Research Products. Ninth Ed. (2003) and the updates thereto, which are each incorporated by reference. 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,
CA), etc. Other 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. 77(3):333, Arakawa et al. (2003) Anal. Biochem. 314(2):206, and Maeda (2003) J. Pharm. Biomed. Anal. 30(6): 1725), and an alpha- methyl-PEG labeling reagent as described in, e.g., U.S. Provisional Patent Application No. 60/428,484, filed on Nov. 22, 2002, which references are each incorporated by reference. Nucleic acid labeling is also described further below. In some embodiments, labeling is achieved using synthetic nucleotides (e.g., synthetic ribonucleotides, etc.) and/or recombinant phycoerythrin (PE).
In addition, whether 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 Quenchers™ or BHQ™, which are commercially available from Biosearch Technologies, Inc. (Novato, CA, USA).
To further illustrate, essentially any nucleic acid (and virtually any labeled nucleic acid, whether standard or non-standard) 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.
In certain embodiments, modified nucleotides are included in probes and primers. To illustrate, 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. Exemplary 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-deaza-dG, N4-ethyl-dC, N6-methyl-dA, etc. To further illustrate, other examples of modified oligonucleotides include those having one or more LNA™ 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
DETECTION OF NUCLEIC ACIDS IN COMPLEX BIOLOGICAL SAMPLES," issued October 16, 2001 to Skouv, and U.S. Pat. Application Pub. No. 2003/0092905, entitled "SYNTHESIS OF [2.2.I]BICYCLO NUCLEOSIDES," by Kochkine et al. that published May 15, 2003, which are each incorporated by reference. Oligonucleotides comprising LNA™ monomers are commercially available through, e.g., Exiqon A/S (Vedbaek, DK). Additional oligonucleotide modifications are referred to herein, including in the definitions provided above.
ARRAY FORMATS
In certain embodiments, oligonucleotide probes designed to hybridize with target nucleic acids are covalently or noncovalently attached to solid supports. In these embodiments, labeled amplicons derived from patient samples are typically contacted with these solid support-bound probes to effect hybridization and detection. In other embodiments, amplicons are attached to solid supports and contacted with labeled probes. Optionally, antibodies, aptamers, or other probe biomolecules utilized in a given assay are similarly attached to solid supports. Essentially any substrate material can be adapted for use as a solid support. In certain embodiments, for example, 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. In some embodiments, 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. Many of these membranous materials are widely available from various commercial suppliers, such as, PJ. Cobert Associates, Inc. (St. Louis, MO, USA), Millipore Corporation (Bedford, MA, USA), or the like. Other 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. 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 Fc 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 β-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.
A number of array systems have been described and can be adapted for use in the detection of target microbial nucleic acids. Aspects of array construction and use are also described in, e.g., Sapolsky et al. (1999) "High-throughput polymorphism screening and genotyping with high-density oligonucleotide arrays" Genetic Analysis: Biomolecular Enfiineering 14:187-192, Lockhart (1998) "Mutant yeast on drugs" Nature Medicine 4:1235-1236, Fodor (1997) "Genes, Chips and the Human Genome" FASEB Journal 1 1 :A879, Fodor (1997) "Massively Parallel Genomics" Science 277: 393-395, and Chee et al. (1996) "Accessing Genetic Information with High-Density DNA Arrays" Science 274:610-614, all of which are incorporated by reference. NUCLEIC ACID HYBRIDIZATION
The length of complementary region or sequence between primer or probes and their binding partners (e.g., target nucleic acids) 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. Although other lengths are optionally utilized, 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. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993), supra, and in Hames and Higgins 1 and Hames and Higgins 2, supra. For purposes of the present invention, generally, "highly stringent" hybridization and wash conditions are selected to be about 5° C or less lower than the thermal melting point (Tm) 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). The Tm 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 T111 for a particular primer or probe.
The Tm 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. Thus, the Tm 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. After hybridization, 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. Higher stringency conditions (e.g., using lower salt and higher temperature that is closer to the hybridization temperature) lowers the background signal, typically with only the specific signal remaining. See, e.g., Rapley et al. (Eds.), Molecular Biomethods Handbook (Humana Press, Inc. 1998), which is incorporated by reference.
Thus, 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.
For example, in determining highly stringent hybridization and wash conditions, 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. For example, 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 least1A 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.
NUCLEIC ACID AMPLIFICATION
In some embodiments, 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. 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. As an exemplary embodiment, 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. An advantage of using target-specific primers in the RT reaction is that only the desired sequences are converted into a PCR template. Superfluous primers or cDNA products are generally not carried into subsequent PCR amplifications. In another embodiment, RNA targets are reverse transcribed using 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. In other embodiments, transcription-based amplification systems (TAS) are used, such as that first described by Kwoh et al. (Proc. Natl. Acad. Sci. (1989)
86(4): 1173-7), or isothermal transcription-based systems such as 3SR (Self- Sustained Sequence Replication; Guatelli et al. (1990) Proc. Natl. Acad. Sci. 87:1874-1878) or NASBA (nucleic acid sequence based amplification; Kievits et al. (1991) J Virol Methods. 35(3):273-86), which are each incorporated by reference. In these methods, the 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. The resulting cDNA products can then serve as templates for multiple rounds of transcription by the appropriate 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. In other exemplary embodiments, amplification is accomplished by used of the ligase chain reaction (LCR), disclosed in European Patent Application No. 320,308
(Backman and Wang), or by the ligase detection reaction (LDR), disclosed in U.S. Patent No. 4,883,750 (Whiteley et al.), which are each incorporated by reference. In LCR, 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. In this variation, 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.
In further embodiments, several methods generally known in the art would be suitable methods of amplification. Some additional examples include, but are not limited to, 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
Chromatography: Template Chromatography of Nucleic Acids and Proteins, Chromatographic Science Series, #27, Marcel Dekker (1984), both of which are incorporated by reference. In certain embodiments, amplicons are not purified prior to detection, such as when amplicons are detected simultaneous with amplification. DATA COLLECTION
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. For example, certain types of labels allow resolution of two species of the same mass through deconvolution of the data. Non-size based differentiation methods (such as deconvolution of data from overlapping signals generated by two different fluorophores) allow pooling of a plurality of multiplexed reactions to further increase throughput.
Separation Methods 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.
In some embodiments, sample analysis includes the use of mass spectrometry. Several modes of separation that determine mass are possible, including Time-of- Flight (TOF), Fourier Transform Mass Spectrometry (FTMS), and quadruple mass . spectrometry. Possible methods of ionization include Matrix-Assisted Laser Desorption and Ionization (MALDI) or Electrospray Ionization (ESI). A preferred embodiment for the uses described in this invention is MALDI-TOF (Wu, 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. In preparing the sample for analysis, 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.
In another embodiment, 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. When combined with confocal imaging methods, these methods provide sensitivity in the range of attomoles, comparable to the sensitivity of radioactive sequencing methods. The use of 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. In many microcapillary electrophoresis methods, the capillaries are filled with an appropriate separation/sieving matrix. Several 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. Generally, 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. For example, 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. For example, 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' LabChip™ 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.
Other embodiments are generally known in the art for separating PCR amplification products by electrophoresis through gel matrices. Examples include polyacrylamide, agarose-acrylamide, or agarose gel electrophoresis, using standard methods (Sambrook, supra).
Alternatively, 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.
Many specialized techniques have been developed for their application including methods utilizing liquid chromatography or HPLC (Katz and Dong (1990) BioTechniques 8(5):546-55; Gaus et al. (1993) J. Immunol. Methods 158:229-236). In yet another embodiment, cDNA products are captured by their affinity for certain substrates, or other incorporated binding properties. For example, 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. Many types of 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. plates, dishes, wells, flasks, dipsticks, membranes), or chromatographic materials (e.g. fibers, gels, screens). 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 LabChip™ microfluidic devices made by Caliper Lifesciences Corp. Another technology developed by
Nanogen, Inc. (San Diego, CA), utilizes microelectronics to move and concentrate biological molecules on a semiconductor microchip. The microfluidics platforms developed at Orchid Biosciences, Inc. (Princeton, NJ), including the Chemtel™ Chip, which provides for parallel processing of hundreds of reactions, can also be used in certain embodiments. These microfluidic platforms require only nanoliter sample volumes, in contrast to the microliter volumes required by other conventional separation technologies.
Some of the processes usually involved in genetic analysis have been miniaturized using microfluidic devices. For example, PCT publication WO 94/05414 reports an integrated micro-PCR apparatus for collection and amplification of nucleic acids from a specimen. U.S. Patent Nos. 5,304,487 (Wilding et al.) and 5,296,375 (Kricka et al.) discuss devices for collection and analysis of cell-containing samples. U.S. Patent No. 5,856,174 (Lipshutz et al.) describes an apparatus that combines the various processing and analytical operations involved in nucleic acid analysis. Each of these references is incorporated by reference. Additional technologies are also contemplated. For example, Kasianowicz et al. (Proc. Natl. Acad. Sci. USA (1996) 93:13770-13773, which is incorporated by reference) describes the use of ion channel pores in a lipid bilayer membrane for determining the length of polynucleotides. In this system, an electric field is generated by the passage of ions through the pores. Polynucleotide lengths are measured as a transient decrease of ionic current due to blockage of ions passing through the pores by the nucleic acid. The duration of the current decrease was shown to be proportional to polymer length. Such a system can be applied as a size separation platform in certain embodiments of the present invention. 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. With 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. In this embodiment, amplification products from each individual reaction are not physically separated, but are differentiated by hybridizing with a set of probes that are differentially labeled. Alternatively, 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. In this embodiment, 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. For example, 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
Following separation of the different products of a multiplex amplification, 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. Many types of 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. In further embodiments, 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. Several types of 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. Several fluorescent moieties, including Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, carboxyfluorescein, Cascade
Blue, Cy3, Cy5, 6-FAM, Fluorescein, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, ROX, TAMRA, TET, Tetramethylrhodamine, and Texas Red, are generally known in the art and routinely used for identification of discrete nucleic acid species, such as in sequencing reactions. Many of these dyes have emission spectra distinct from one another, enabling deconvolution of data from incompletely resolved samples into individual signals. This allows pooling of separate reactions that are each labeled with a different dye, increasing the throughput during analysis, as described in more detail below. Additional examples of suitable labels are described herein. The signal strength obtained from fluorescent dyes can be enhanced through use of related compounds called energy transfer (ET) fluorescent 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. Use of these coupled-dye systems can significantly amplify fluorescent signal. Examples of ET dyes include the ABI PRISM BigDye terminators, recently commercialized by Perkin-Elmer Corporation (Foster City,
CA, USA) for applications in nucleic acid analysis. These 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. 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. Alternatively, 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. (1998) 26:1996-2000, which is incorporated by reference), describes hybridization of a labeled oligonucleotide probe to its target without physical separation from unhybridized probe. In this method, the probe is labeled with a chemiluminescent molecule that in the unbound form is destroyed by sodium sulfite treatment, but is protected in probes that have hybridized to target sequence.
In other embodiments, 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.
Some preferred compounds that can serve as an IR label include an aromatic nitrile, aromatic alkynes, or aromatic azides. Numerous compounds can serve as an EC label; many are listed in PCT publication WO 97/27327. 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. In this embodiment, 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. Various coupling strategies are possible utilizing well-characterized interactions generally known in the art, such as those between biotin and avidin, an antibody and antigen, or a sugar and lectin. Various types of enzymes can be employed, generating colorimetric, fluorescent, chemiluminescent, phosphorescent, or other types of signals. As an illustration, 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. One of skill in the art can conceive of many possible variations on the different embodiments of detection methods described above.
In some embodiments, it may be desirable prior to detection to separate a subset of amplification products from other components in the reaction, including other products. 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. After 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. In some embodiments, the binding interaction providing for capture may also serve as the mechanism of detection.
Furthermore, the size of an amplification product or products are optionally changed, or "shifted," in order to better resolve the amplification products from other products prior to detection. For example, chemically cleavable primers can be used in the amplification reaction. In this embodiment, 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.
DATA ANALYSIS
For reliably diagnosing leukemia, for example, it is generally desirable to determine the expression of more than one of the markers of the disease. As an exemplary criterion for the choice of markers, 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). In some embodiments, 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. Typically, 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. In certain embodiments, 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, which is incorporated by reference, 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. In some embodiments, 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%. To further illustrate, 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.
In certain embodiments, for example, 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. On the other hand, 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). 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,
Wiley (1998), which are each incorporated by reference.
To further illustrate, 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. Generally, 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). Based on identified distinct gene expression signatures, 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. Based on the training set, a Support Vector Machine (SVM) model can be built using this approach.
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. This prediction approach can be applied both for overall classification (multi-class) and binary classification (diagnosis X => yes or no). For the latter, sensitivity and specificity are optionally calculated, as follows:
Sensitivity = (number of positive samples predicted)/(number of true positive) Specificity = (number of negative samples predicted)/(number of true negatives).
EXAMPLES It is understood that the examples and embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the claimed invention. It is also understood that various modifications or changes in light the examples and embodiments described herein will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
EXAMPLE 1: PATTERN ROBUSTNESS OF DIAGNOSTIC GENE EXPRESSION SIGNATURES
INTRODUCTION
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."
Pre-existing approaches to diagnosing leukemia typically utilize a combination of complementary methods, are expensive and time-consuming, and require expert knowledge. Methods that generally have to be individually combined are cytomoφhology or histomorphology, multiparameter immunophenotyping, cytogenetics, fluorescence in situ hybridization (FISH), and molecular genetics such as polymerase chain reaction-based assays. Moreover, the subclassification of leukemias becomes increasingly important to guide therapy. Tailored drugs can dramatically improve the outcome of distinct subsets of leukemia. 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).12'13 Promising data has been shown on selected cohorts of patients supporting an application of microarray technology for the diagnosis of leukemias."''4'15 With respect to the identification and application of genes for a diagnostic usage, various parameters that may influence gene expression patterns were examined in this analysis.
MATERIAL AND METHODS
Samples were analyzed from n=240 untreated adult leukemia patients at diagnosis.
The diagnostic subclasses can be itemized as follows: AML with t(l 5; 17) (n=42), t(8;21) (n=40), inv(16) (n=49), t(l Iq23)/MLL (n=51), inv(3) (n=22), as well as CML (n=36). All samples were referred to the Laboratory for Leukemia Diagnostics, Munich, Germany between December 1998 and January 2004, and registered in our leukemia database.16 The studies were conducted following the rules of the local internal review board and the tenets of the revised Helsinki protocol. In all patients that were included in this investigation, the diagnosis was performed by the investigators using an individual combination of cytomorphology, cytogenetics, fluorescence in situ hybridization (FISH), immunophenotyping and molecular genetics. At the individual time point of diagnosis mononuclear cells were purified by Ficoll density centrifugation and lyzed (RLT lysis buffer, Qiagen, Hilden, Germany). For subsequent microarray analyses these stabilized lysates had been stored at -800C. Gene expression analyses were performed as previously described.14'15'17*18 MICROARRAY DATA ANALYSIS AND VISUALIZATION
Gene expression data was preprocessed according to Affymetrix' recommendations and analyzed as follows: For each disease entity differentially expressed genes were calculated by means oft-test-statistic (two-sample t-test, unequal variances) in a one-versus-all (OVA) approach. We applied the software package R version
1.7.1 available on the world wide web at r-project.org. To address the multiple testing problem, false discovery rates (FDR) of genes were calculated.19'20 To assess similarity of gene expression patterns hierarchical cluster analyses and principal component analyses were applied.21'2 Transformed gene expression data was analyzed using the GeneMaths XT software from Applied Maths, Belgium
(cluster algorithm: Ward; selected coefficient: Euclidean distance).
RESULTS AND DISCUSSION
Formerly made hypotheses that leukemia-specific chromosomal aberrations translate into dramatic changes on the transcriptional level have now be confirmed by several studies from various diagnostic and research centers.2'7'10'1 ''13'15'18'23"25
In acute myeloid leukemia (AML), 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.2'7'15'18 In acute lymphoblastic leukemia (ALL) gene expression profiling was successfully applied to accurately identify the known prognostically relevant subclasses, including precursor T-cell ALL, E2A-PBX1, TEL-AMLl, MLL gene rearrangements, BCR-ABL, and hyperdiploid karyotypes > 50 chromosomes.1 '' ' In addition, the patterns are reproducible across, e.g., different array platforms
(spotted cDNA versus in situ synthesized oligonucleotides), microarray designs (Affymetrix U95A versus U 133A), and age groups of patients.2'14'18
In this study, the robustness of diagnostic gene expression patterns was analyzed. As a nation-wide reference center for the diagnosis of hematological malignancies, patient samples were received from all over Germany, either from local hospitals or via express mailing. For this reason the samples were heterogeneous with respect to shipment time which might bias RNA quality and subsequent gene expression profiles. Also, when conducting a gene expression profiling study one should consider that varying storage periods of the samples or target preparations at different time points from either bone marrow or peripheral blood specimens might influence the leukemic gene expression signatures.
As an example, the robustness of diagnostic gene expression patterns was approached in this analysis by visualizing gene signatures according to various parameters. In particular, in a first analysis including AML with t(15;17), t(8;21), inv(16), and t(l Iq23)/MLL cases 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. As shown in Figure IA through IF no glaring changes occurred for the various conditions when using a set of differentially expressed genes. For each parameter, AML subtypes with recurrent chromosomal aberrations show homogeneous signatures, which would enable an accurate prediction when applied in a diagnostic setting. Secondly, the handling of the samples, e.g. freezing of cell preparations versus freshly prepared samples, and storage time of the samples at -80°C may vary within a larger profiling study. Also, differences between operators for sample target preparation and microarray washing and scanning procedures might represent interfering factors. There was no indication that these variations in sample handling and operators influence a diagnostic signature. As shown in Figure 2, some samples were transferred to another laboratory in order to assess the influence of sample preparation operators. Here, two patient cohorts were analyzed, i.e. AML with t(15;17) and inv(16). Differentially expressed genes between these two entities were identified based on the expression profiles according to the preparation of operator 1 , operator 2 or including all samples from both operators, respectively (Figure 2A,B,C,D). No influence on the robustness of the signature was seen. Moreover, expression profiles generated from a freshly prepared sample versus samples that had been frozen as cell lysates for several days at -80°C were also analyzed. Figure 2E shows that different storage times also had no impact on the signature. This analysis also included preparations at three different time points from one patient, i.e. immediate target preparation, and preparations after 24h and 48h storage at room temperature, respectively. In a final step, the two operators working in different laboratories prepared six samples in duplicates. As shown in Figures 2E and F, for each sample the four individual profiles cluster near to each other indicating the robustness of the sample preparation protocol, microarray platform, and resulting gene expression signature.
In conclusion, this analysis illustrated that for a subset of leukemias, i.e. AML with recurrent chromosomal aberrations, gene expression profiling is applicable in a diagnostic setting considering various influences. Using a set of n=287 differentially expressed genes, four genetically defined AML subtypes were robustly identified under various influencing conditions. In addition, preparations from different operators, and different handling procedures of the samples did not impair robustness of diagnostic gene expression patterns.
EXAMPLE 2: ADDITIONAL ANALYSIS INTRODUCTION
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.
Several types of microarray platforms exist, 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.
MATERIAL AND METHODS
Gene expression signatures had been identified to discriminate different leukemia types using Affymetrix DNA-oligonucleotide microarrays HG-Ul 33A and HG-
U133B. These predefined diagnostic gene expression signatures were tested in a study using the HG-Ul 33 Plus 2.0 microarray and the GeneChip® 2.0 Dx diagnostics platform. The study included prospectively collected samples from leukemia patients at diagnosis. More than 90% of the samples had been sent consecutively to our laboratory between January and July 2004. The median shipment time was 24 hours. The median percentage of blasts in acute leukemias was 80%. A total number of 411 target preparations was performed and resulted in 400 hybridization cocktails (97.3% success rate). The sample target preparation protocol included the current assay recommendations for nucleic acid quantification and cleanup, IVT labeling and hybridization and allows to prepare samples in less than 24 hours. The microarrays were automatically washed and stained with the fluidics station. A scanner with removable 48-array autoloader carousel maximized array throughput was utilized in the analysis. For statistical comparison 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.
RESULTS Overall 378/400 (94.5%) of the independent test samples were correctly classified.
Some of the misclassifications could be explained by the underlying biology of the respective samples. EXAMPLE 3: GENERAL MATERIALS. METHODS AND DEFINITIONS OF FUNCTIONAL ANNOTATIONS
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.
AFFYMETRIX PROBESET ANNOTATION
All annotation data of GeneChip® arrays are extracted from the NetAffx™ Analysis Center (internet website: www.affymetrix.com). Files for Ul 33 set arrays, including U133A and U133B microarrays are derived from the June 2003 release. The original publication refers to: Liu et al. (2003) "NetAffx: Affymetrix probe sets and annotations," Nucleic Acids Res. 31(1^:82-6, which is incorporated by reference.
The 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.
DATA FIELDS
In the following section, the content of each field of the data files is described. 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:
1. GeneChip Array Information
2. Probe Design Information 3. Public Domain and Genomic References
1. GeneChip Array Information HG-U 133 ProbeSet ID:
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). 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. For 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.
Examples are: GenBank®, RefSeq, UniGene, TIGR (annotations from The Institute for Genomic Research).
2. Public Domain and Genomic References Most of the data in this section is from the LocusLink and UniGene databases, and are annotations of the reference sequence on which the probe set is modeled.
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.
MapLocation:
The map location describes the chromosomal location when one is available. Unigene Accession: UniGene accession number and cluster type. Cluster type can be "full length" or
"est", or "— " if unknown.
LocusLink: This information represents the LocusLink accession number.
Full Length Ref. Sequences Indicates the references to multiple sequences in RefSeq. The field contains the ID and description for each entry, and there can be multiple entries per probeSet.
EXAMPLE 4: SAMPLE PREPARATION. PROCESSING AND DATA ANALYSIS
Microarray analyses were performed utilizing the GeneChip® System (Affymetrix,
Santa Clara, USA). 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,
Germany), frozen, and stored at -80°C from 1 week to 38 months. For gene expression profiling cell lysates of the leukemia samples were thawed, homogenized (QIAshredder, Qiagen), and total RNA was extracted (RNeasy Mini Kit, Qiagen). Subsequently, 5-10 μg total RNA isolated from 1 x 107 cells was used as starting material for cDNA synthesis with oligo[(dT)24T7promotor]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). After quantification by spectrophotometric measurements and 260/280 absorbance values assessment for quality control of the purified cRNA (RNeasy Mini Kit, Qiagen), 15 μg 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 μL 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.
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While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one' skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes.