Compositions for the detection of blood cell and immunological response gene expression

[0002] The present invention relates to a composition comprising a plurality of polynucleotide probes for use in research and diagnostic applications.

[0003] DNA-based arrays can provide a simple way to explore the expression of a single polymorphic gene or a large number of genes. When the expression of a single gene is explored, DNA-based arrays are employed to detect the expression of specific gene variants. For example, a p53 tumor suppressor gene array is used to determine whether individuals are carrying mutations that predispose them to cancer. The array has over 50,000 DNA probes to analyze more than 400 distinct mutations of p53. A cytochrome p450 gene array is useful to determine whether individuals have one of a number of specific mutations that could result in increased drug metabolism, drug resistance or drug toxicity.

[0004] DNA-based array technology is especially relevant for the rapid screening of expression of a large number of genes. There is a growing awareness that gene expression is affected in a global fashion. A genetic predisposition, disease or therapeutic treatment may affect, directly or indirectly, the expression of a large number of genes. In some cases the interactions may be expected, such as where the genes are part of the same signaling pathway. In other cases, such as when the genes participate in separate signaling pathways, the interactions may be totally unexpected. Therefore, DNA-based arrays can be used to investigate how genetic predisposition, disease, or therapeutic treatment affects the expression of a large number of genes.

[0005] cDNA-based arrays have been used in discovery and analysis of inflammatory disease related genes (Heller et al. (1997) Proc. Natl. Acad. Sci USA94: 2150-2155). A first type of array was employed to characterize the expression patterns of a class of 96 genes coding for polypeptides known to be involved in rheumatoid arthritis. This array contained preselected probes for the 96 genes. A second type of array was used to investigate gene expression patterns characteristic of blood cells. This array contained probes for 1,000 human genes randomly selected from a human blood cell cDNA library.

[0006] Current cDNA-based arrays suffer from a variety of limitations. One is the first type of array can only detect the expression patterns of a limited number of genes already associated with a disease. The expression of other, yet to be identified, relevant genes is not detected. Another is the second type of array contains probes for genes that have very little to do with the regulation of inflammation. Also, high abundance genes are likely to be over represented and low abundance genes are likely to be under represented. The present invention provides a way to overcome such limitations.

[0007] In one aspect, the present invention provides a composition comprising a plurality of polynucleotide probes, wherein each of said polynucleotide probes comprises at least a portion of a gene implicated in blood cell biology. The plurality of polynucleotide probes can be selected from I) first polynucleotide probes, wherein each of said first polynucleotide probes comprises at least a portion of a gene differentially expressed in an immunological response; II) second polynucleotide probes, wherein each of said second polynucleotide probes comprises at least a portion of a gene abundantly expressed in an immunological response; III) third polynucleotide probes, wherein each of said third polynucleotide probes comprises at least a portion of a gene coding for a polypeptide known to regulate blood cell biology; and IV) combinations of first, second or third polynucleotide probes.

[0008] Preferably, the plurality of polynucleotide probes comprises: I) first polynucleotide probes, wherein each of said first polynucleotide probes comprises at least a portion of a gene differentially expressed in an immunological response; II) second polynucleotide probes, wherein each of said second polynucleotide probes comprises at least a portion of a gene abundantly expressed in an immunological response; and III) third polynucleotide probes, wherein each of said third polynucleotide probes comprises at least a portion of a gene coding for a polypeptide known to regulate blood cell biology.

[0009] Generally, first polynucleotide probes are selected by a) preparing at least one first target transcript profile from a first biological sample selected from the group consisting of hematopoietic cells and inflamed tissue and at least one first subtraction transcript profile from a noninflamed, nonhematopoietic biological sample; b) subtracting said first subtraction transcript profile from said first target profile to detect a plurality of genes that are differentially expressed in an immunological response; and c) identifying one of said detected genes that are differentially expressed in an immunological response. Second polynucleotide probes are selected by a) preparing at least one second target transcript profile from a second biological sample selected from the group consisting of hematopoietic cells and inflamed tissue to detect genes that are abundantly expressed in said second biological sample; and b) identifying one of said detected genes that are abundantly expressed. Third polynucleotide probes are selected by a third method comprising identifying a gene coding for a polypeptide with a known function in immunological responses.

[0010] In one preferred embodiment, the composition comprises a plurality of polynucleotide probes, wherein each polynucleotide probe comprises at least a portion of a sequence selected from the group consisting of SEQ ID Nos: 1-1508. In a second preferred embodiment, the composition comprises a plurality of polynucleotide probes comprising at least a portion of at least about 1000 of the sequences of SEQ ID Nos: 1-1508. In yet another embodiment, the composition comprises a plurality of polynucleotide probes wherein said polynucleotide probes comprise at least a portion of substantially all the sequences of SEQ ID Nos: 1-1508. The polynucleotide probes can be cDNAs, clone DNAs and the like.

[0011] The composition is particularly useful as hybridizable array elements in a microarray for monitoring the expression of a plurality of target polynucleotides. The microarray comprises a substrate and the hybridizable array elements. The microarray can be used, for example, in the diagnosis and treatment of an immunopathology.

[0012] In another aspect, the present invention provides an expression profile that can reflect the levels of a plurality of target polynucleotides in a sample. The expression profile comprises a microarray and a plurality of detectable complexes. Each detectable complex is formed by hybridization of at least one of said target polynucletodies to at least one of said polynucleotide probes and further comprises a labeling moiety for detection.

[0013] In yet another aspect, the present invention provides a method for identifying a plurality of polynucleotide probes. The method comprises selecting I) first polynucleotide probes, wherein each of said first polynucleotide probes comprises at least a portion of a gene differentially expressed in an immunological response; II) second polynucleotide probes, wherein each of said second polynucleotide probes comprises at least a portion of a gene abundantly expressed in an immunological response; and III) third polynucleotide probes, wherein each of said third polynucleotide probes comprises at least a portion of a gene coding for a polypeptide known to regulate blood cell biology.

[0014] A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0015] The Sequence Listing is a compilation of nucleotide sequences obtained by sequencing clone inserts (isolates) of different cDNA libraries. Each sequence is identified by a sequence identification number (SEQ ID No:), by the clone number from which it was obtained and by the cDNA library from which the sequence was obtained.

[0016] Table 1 is a list of the sequences according to the SEQ ID Nos:. For SEQ ID Nos: 1-854 (homologous to GenBank polypeptide or nucleotide sequences), the first column contains Incyte clone numbers. The second column contains relevant GenBank Identification numbers, if any. The last column contains an annotation associated with the referenced GenBank identification numbers. For SEQ ID Nos: 855-1508 (exact matches to GenBank) the first column contains the GenBank identification number and the second column contains an annotation associated with the referenced GenBank identification number.

[0017] Table 2 is a list of the cDNA libraries and a description of the preparation of the cDNA libraries.

[0018] FIG. 1 is an image derived from an experiment where gene expression of untreated THP1 cells is investigated using a microarray comprising cDNA polynucleotide probes.

[0019] FIG. 2 is an image derived from an experiment where gene expression of treated THP cells is investigated using a microarray comprising cDNA polynucleotides.

[0020] The term “microarray” refers to an ordered arrangement of hybridizable array elements. The array elements are arranged so that there are preferably at least one or more different array elements, more preferably at least 100 array elements, and most preferably at least 1,000 array elements, on a 1 cm²substrate surface. The maximum number of array elements is unlimited, but is at least 100,000 array elements. Furthermore, the hybridization signal from each of the array elements is individually distinguishable. In a preferred embodiment, the array elements comprise polynucleotide probes.

[0021] A “polynucleotide” refers to a chain of nucleotides. Preferably, the chain has from about 100 to 10,000 nucleotides, more preferably from about 150 to 3,500 nucleotides. The term “probe” refers to a polynucleotide sequence capable of hybridizing with a target sequence to form a polynucleotide probe/target complex. A “target polynucleotide” refers to a chain of nucleotides to which a polynucleotide probe can hybridize by base pairing. In some instances, the sequences will be complementary (no mismatches). In other instances, there may be a 10%, mismatch.

[0022] A “plurality” refers preferably to a group of at least one or more members, more preferably to a group of at least about 100, and even more preferably to a group of at least about 1,000, members. The maximum number of members is unlimited, but is at least about 100,000 members.

[0023] A “portion” means a stretch of at least about 100 consecutive nucleotides. A “portion” can also mean a stretch of at least 100 consecutive nucleotides that contains one or more deletions, insertions or substitutions. A “portion” can also mean the whole coding sequence of a gene. Preferred portions are those that lack secondary structure as identified by using computer software programs such as OLIGO 4.06 Primer Analysis is Software, (National Biosciences, Plymouth, Minn.) LASERGENE software (DNASTAR, Madison Wis., MACDASIS software (Hitachi Software Engineering Co., Ltd. South San Francisco, Calif.) and the like.

[0024] The term “gene” or “genes” refers to the partial or complete coding sequence of a gene. The phrase “genes implicated in blood cell biology” refers to genes that code for polypeptides that are known to regulate blood cell biology and genes of unknown function which are differentially or abundantly expressed in hematopoiesis or immunological responses and include those listed in the Sequence Listing and in Table 1.

[0025] The phrase “differentially expressed gene” refers to a gene whose abundance in a target transcript profile is preferably at least about 1.5×higher, more preferably about 2×higher, than that in a subtraction transcript profile. The phrase also refers to genes that are not detectable in the subtraction transcript profile but are preferably at levels of at least about 2 copies per cell, more preferably at least about 3 copies per cell, in the target transcript profile. “Abundantly expressed gene” refers to a gene which represents preferably at least about 0.01% of the transcripts in a transcript profile.

[0026] As used herein, the profile of transcripts which reflect gene expression in a particular tissue, at a particular time, is defined as a “transcript profile”. Such profiles can be generated by naming, matching, and counting all copies of related clone inserts and arranging them in order of abundance. A “target transcript profile” refers to a profile derived from a biological sample that contains transcripts of interest along side transcripts which are not of interest. A “subtraction transcript profile” refers to a profile derived from a biological sample that contains predominantly transcripts that are not of interest.

[0027] The phrase “blood cell biology” encompasses hematopoeisis and all variety of immunological responses, including T cell and B cell activation, monocyte activation, and the like, and immunopathology.

[0028] “Hematopoeisis” refers to the process of blood cell growth and differentiation. “Immunological response” refers to responses elicited from blood cells including normal and immunopathological responses.

[0029] The phrase “genes coding for a polypeptide known to regulate blood cell biology” refers to genes whose known function is related to immunological responses, such as cytokines, chemokines, growth factors, transcription factors, leukotrienes, cell surface receptors, phosphatases and the like.

[0030] The term “hematopoietic cells” include erythrocytes, neutrophils, eosinophil, basophils, mast cells, megakaryocytes, platelets, monocytes, macrophages, dendritic cells, T lymphocytes, B lymphocytes, natural killer cells and the like. Furthermore, the term includes cells from tissues such as spleen, thymus, adenoid gland, fetal liver tissue and the like.

[0031] The present invention provides a composition comprising a plurality of polynucleotide probes comprising at least a portion of genes implicated in blood cell biology. Preferably, the polynucleotide probes comprise at least a portion of one or more of the sequences (SEQ ID Nos: 1-1508) presented in the Sequence Listing. In one preferred embodiment, the composition comprises a plurality of polynucleotide probes, wherein each polynucleotide probe comprises at least a portion of a sequence selected from the group consisting of SEQ ID Nos: 1-1508. In a second preferred embodiment, the composition comprises a plurality of polynucleotide probes comprising at least a portion of at least about 1000 of the sequences of SEQ ID Nos: 1-1508. In yet another embodiment, the composition comprises a plurality of polynucleotide probes wherein said polynucleotide probes comprise at least a portion of substantially all the sequences of SEQ ID Nos: 1-1508.

[0032] The composition is particularly useful when it is used as hybridizable array elements in a microarray. Such a microarray can be employed to monitor the expression of genes of unknown function, but which are differentially or abundantly expressed in an immunological response or an immunopathology. In addition, the microarray can be used to monitor the expression of genes with a known function in blood cell biology.

[0033] The microarray can be used for large scale genetic or gene expression analysis of a large number of target polynucleotides. The microarray can be used in the diagnosis of diseases and in the monitoring of treatments where altered expression of genes implicated in blood cell biology cause disease, such as cancer, an immunopathology and the like. The microarray can also be used to investigate an individual's predisposition to a disease, such as cancer, an immunopathology and the like. Furthermore, the microarray can be employed to investigate cellular responses, such as stress responses, apoptosis, cell proliferation and the like.

[0034] When the composition of the invention is employed as hybridizable array elements in a microarray, the array elements are organized in an ordered fashion so that each element is present at a specified location on the substrate. Because the array elements are at specified locations on the substrate, the hybridization patterns and intensities (which together create a unique expression profile) can be interpreted in terms of expression levels of particular genes and can be correlated with a particular disease or condition or treatment.

[0035] The composition comprising a plurality of polynucleotide probes can also be used to purify a subpopulation of mRNAs, cDNAs, genomic fragments and the like, in a sample. Typically, samples will include the target polynucleotides of interest and other nucleic acids which may enhance the hybridization background in the sample. Therefore it may be advantageous to remove these nucleic acids. One method for removing the additional nucleic acids is by hybridizing the sample containing target polynucleotides with immobilized polynucleotide probes under hybridizing conditions. Those nucleic acids that do not hybridize to the polynucleotide probes are washed away. At a later point, the immobilized target polynucleotide probes can be released in the form of purified target polynucleotides.

[0036] Method for Selecting Polynucleotide Probes

[0037] This section describes the selection of probe sequences for the plurality of polynucleotide probes. The probe sequences are selected by identifying genes coding for polypeptides with a known function in immunological responses or genes which are abundantly or differentially expressed in specific biological samples. Since some of the probe sequences are identified solely based on expression levels, it is not essential to know a priori the function of a particular gene in blood cell biology.

[0038] The selection method is based, in part, on expression sequence tag (EST) analysis. EST analysis entails sequencing, in whole or in part, isolated clone inserts from a complementary DNA (cDNA) library, clustering overlapping sequences and determining the clustered sequences' frequency in the cDNA library.

[0039] ESTs are sequenced by methods well known in the art. The methods can employ such enzymes as the Klenow fragment of DNA polymerase I, Taq polymerase, thermostable T7 polymerase, or combinations of polymerases and proofreading exonucleases. Preferably, the process is automated. ESTs derived from the same transcript can be combined to form a cluster of ESTs. Clusters are formed by identifying overlapping EST sequences and assembling the ESTs. A nucleic acid fragment assembly tool, such as the PHRAP tool (WashU-Merck) and the GELVIEW Fragment Assembly system (Genetics Computer Group, Madison, Wis.), can be used for this purpose. Clones can be arranged in clusters in descending order of abundance. The minimum number of clones necessary to constitute a cluster is two.

[0040] After assembling EST clusters, a transcript profile for a particular biological sample is generated and the frequency or abundance of a given EST cluster can be determined. The frequency of an EST cluster in a clone population is correlated to the level of expression of a particular gene. By this process those genes that are abundantly expressed in a biological sample can be identified.

[0041] Furthermore, EST analysis can be employed to identify genes that are differentially expressed in one biological sample (from which a target cDNA library and a target transcript profile are derived) but not in another biological sample (from which a subtraction cDNA library and a subtraction transcript profile are derived). For this purpose, transcript profiles from both biological samples are generated compared. By comparing transcript profiles, those genes that are differentially expressed in a target biological sample can be identified.

[0042] With a large enough number of transcript profiles derived from different biological samples, a statistically significant correlation can emerge between cell and tissue source information, such as disease states, treatment outcomes, exposure to various environmental factors or genotypes, and the expression levels of particular genes or groups of genes. Comparisons between transcript profiles of different cells or tissues or of the same cells or tissues under different conditions can be used to discern differences in transcriptional activities. For example, a transcript profile can show differences occurring between two different tissues, such as liver and prostate; between normal and diseased tissue, such as normal and prostate tumor or between untreated and treated tissues, such as prostate tumor and irradiated prostate tumor.

[0043] The biological samples from which transcript profiles are derived can be from a variety of sources. For purposes of this invention, since the intent is to select polynucleotide probes useful for investigating gene expression as it relates to blood cell biology, biological samples include those derived from hematopoietic and inflamed samples and nonhematopoietic, noninflamed biological samples.

[0044] In particular, where probe sequences are derived from genes differentially expressed in an immunological response, the transcript profiles of hematopoietic cells or tissues associated with an immunological response (normal or inflamed) are compared to those of noninflamed nonhematopoietic samples. Examples of hematopoietic cells or tissues associated with an immunological response include inflamed adenoid, bone marrow, macrophages, lymphocytes, granulocytes, spleen, tonsil, eosinophil, asthmatic lung tissue, diabetic pancreas, colon tissue derived from an individual suffering from Crohn's disease and the like. Examples of noninflamed nonhematopoietic tissue include fibroblasts, keratinocytes, fetal lung, brain, melanocytes and the like. Only those genes that are differentially expressed, i.e., the transcript levels are preferably at least about 1.5×higher, more preferably at least about 2×higher, in hematopoietic sample than that in the nonhematopoietic sample, are selected. Additionally, genes that are not detectable in the nonhematopoietic sample but which have transcript levels of preferably at least about two (2) copies per cell, more preferably at least about three (3) copies per cell, in the hematopoietic sample are selected.

[0045] Where probe sequences are derived from genes that are abundantly expressed in an immunological response, the transcript profiles of hematopoietic cells or tissues associated with an inflammatory process are obtained. Only those genes whose transcripts represent preferably at least 0.01% of the transcripts in a biological sample are selected.

[0046] For purposes of this invention, transcript profile comparisions can be obtained by methods well known to those skilled in the art. Transcript levels and profiles can be obtained and compared, for example, by a differential gene expression assay based on a quantitative hybridization of arrayed cDNA clones (Nguyen, et al. (1995) Genomics29: 207-216), based on the serial analysis of gene expression (SAGE) technology (Velculescu et al. (1995) Science270: 484-487), based on the polymerase chain reaction (Peng et al. (1992) Science257: 967-971, Prashar et al. (1996) Proc. Natl. Acad. Sci. USA93: 659-663), by a differential amplification protocol (Van Gelder et al. 5,545,522), or based on electronic analysis, such as the Lifeseq® Transcript Imaging tool (Incyte Pharmaceuticals, Palo Alto, Calif. hereinafter Incyte) or the GeneCalling and Quantitative Expression Analysis technology (Curagen, New Haven, Conn.) Comparisons (subtractions) between two of more transcript profiles are preferably performed electronically using the Lifeseq® Multiple Transcript Subsetting tool (Incyte).

[0047] Selection Protocols The method of selecting polynucleotide probe sequences is based on three selection protocols. The polynucleotide probes of the composition can be selected by employing any one of these three selection protocols, the combination of any two of these protocols, or all the protocols.

[0048] A first selection protocol (I) can provide for first polynucleotide probes derived from genes differentially expressed in an immunological response. A number of target cDNA libraries are prepared from first biological samples. The first biological sample can be hematopoietic cells or tissues associated with an immunological response, such as inflamed tissues. Preferably at least one cDNA library, more preferably at least four cDNA libraries, are selected from hematopoietic cells or tissues associated with an immunological response. First target transcript profiles are generated from each of the libraries. Transcript profiles can be combined to obtain an averaged transcript profile. An averaged transcript profile entails adding up the transcript abundances for each transcript from each biological sample and then dividing summed up transcript abundances by the total number of biological samples.

[0049] A number of subtraction cDNA libraries are prepared from biological samples that are noninflamed and nonhematopoietic. Preferably at least one cDNA library, more preferably at least four cDNA libraries, are selected from noninflamed nonhematopoietic biological samples. First subtraction transcript profiles are generated from these cDNA libraries. Preferably, these transcript profiles are combined to obtain an averaged transcript profile.

[0050] In one embodiment, the averaged transcript image from the subtraction cDNA libraries is subtracted from each target cDNA library. In another embodiment, the averaged transcript image from the subtraction cDNA libraries is subtracted from an averaged transcript image of the target cDNA libraries.

[0051] In either case, a transcript profile is obtained showing the genes that are differentially expressed in biological samples consisting of hematopoietic cells or tissues associated with an immunological response rather than with noninflamed and nonhematopoietic biological samples. In one embodiment, the top 100 most abundant transcripts, more preferably the top 40 most abundant transcripts, are selected to generate first polynucleotide probes. In a second embodiment, all upregulated transcripts are selected. By upregulated is meant that the genes are not detectable in the subtraction transcript profile but are preferably at levels of at least about 2 copies per cell, more preferably at least about 3 copies per cell, in the target transcript profile.

[0052] A second selection protocol (II) can provide for second polynucleotide probes derived from genes abundantly expressed in an immunological-response. A number of target cDNA libraries are prepared from second biological samples. The second biological sample can be hematopoietic cells or tissues associated with an inflammatory process (normal or diseased). Preferably at least one cDNA library, more preferably at least four cDNA libraries, are selected from hematopoietic cells or tissues associated with an inflammatory process. Third transcript profiles are generated from such libraries. The transcripts are ranked according to abundance. Those transcripts that are most abundant are selected. Preferably the top 100 most abundant transcripts are selected from the remaining transcripts, more preferably the top 40 most abundant transcripts, including the top 20 novel sequences (i.e., not in a public database), can be selected to generate second polynucleotide probes.

[0053] In a third selection protocol (III) the literature is surveyed and sequences in GenBank and Lifeseq® database (Incyte) screened to identify genes coding for polypeptides whose function is related to immunological responses. These genes can be selected to generate third polynucleotide probes.

[0054] The resulting composition can comprise polynucleotide probes that are not redundant, i.e., there is no more than one polynucleotide probe to represent a particular gene. Alternatively, the composition can contain polynucleotide probes that are redundant, i.e., a gene is represented by more than one polynucleotide probe.

[0055] The selected polynucleotide probes may be manipulated further to optimize the performance of the polynucleotide probes as hybridization probes.

[0056] Some probes may not hybridize effectively under hybridization conditions due to secondary structure. To optimize probe hybridization, the probe sequences are examined using a computer algorithm to identify portions of genes without potential secondary structure. Such computer algorithms are well known in the art such as OLIGO 4.06 Primer Analysis Software (National Biosciences) or LASERGENE software (DNASTAR). These programs can search nucleotide sequences to identify stem loop structures and tandem repeats and to analyze G+C content of the sequence (those sequences with a G+C content greater than 60% are excluded). Alternatively, the probes can be optimized by trial and error. Experiments can be performed to determine whether probes and complementary target polynucleotides hybridize optimally under experimental conditions.

[0057] Where the number of different polynucleotide probes is desired to be greatest, the probe sequences are extended to assure that different polynucleotide probes are not derived from the same gene, i.e., the polynucleotide probes are not redundant. The probe sequences may be extended utilizing the partial nucleotide sequences derived from EST sequencing by employing various methods known in the art. For example, one method which may be employed, “restriction-site” PCR, uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, G. (1993) PCR Methods Applic. 2: 318-322).

[0058] Polynucleotide Probes

[0059] This section describes the polynucleotide probes. The polynucleotide probes can be DNA or RNA, or any RNA-like or DNA-like material, such as peptide nucleic acids, branched DNAs and the like. The polynucleotide probes can be sense or antisense polynucleotide probes. Where target polynucleotides are double stranded, the probes may be either sense or antisense strands. Where the target polynucleotides are single stranded, the nucleotide probes are complementary single strands.

[0060] In one embodiment, the polynucleotide probes are cDNAs. The size of the DNA sequence of interest may vary, and is preferably from about 100 to 10,000 nucleotides, more preferably about from 150 to 3,500 nucleotides.

[0061] In a second embodiment, the polynucleotide probes are clone DNAs. In this case, the size of the DNA sequence of interest, i.e., the insert sequence excluding the vector DNA, may vary from 100 to 10,000 nucleotides, more preferably from 150 to 3,500 nucleotides.

[0062] The polynucleotide probes can be prepared by a variety of synthetic or enzymatic schemes which are well known in the art. The probes can be synthesized, in whole or in part, using chemical methods well known in the art. (Caruthers et al. (1980) Nucleic. Acids Symp. Ser. (2) 215-233). Alternatively, the probes can be generated, in whole or in part, enzymatically.

[0063] Nucleotide analogues can be incorporated into the polynucleotide probes by methods well known in the art. The only requirement is that the incorporated nucleotide analogues must serve to base pair with target polynucleotide sequences. For example, certain guanine nucleotides can be substituted with hypoxanthine which base pairs with cytosine residues. However, these base pairs are less stable than those between guanine and cytosine. Alternatively, adenine nucleotides can be substituted with 2,6-diaminopurine which can form stronger base pairs than those between adenine and-thymidine.

[0064] Additionally, the polynucleotide probes can include nucleotides that have been derivatized chemically or enzymatically. Typical chemical modifications include derivatization with acyl, alkyl, aryl or amino groups.

[0065] The polynucleotide probes can be immobilized on a substrate. Preferred substrates are any suitable rigid or semirigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which the polynucleotide probes are bound. Preferably, the substrates are optically transparent.

[0066] Probes can be synthesized, in whole or in part, on the surface of a substrate by using a chemical coupling procedure and a piezoelectric printing apparatus, such as that described in PCT publication WO95/251116 (Baldeschweiler et al.). Alternatively, the probe can be synthesized using a self-addressable electronic device that controls when reagents are added (Heller et al. U.S. Pat. No. 5,605,662) or by photolysis using imaging fibers for light delivery (Healey et al. (1995) Science269: 1078-80).

[0067] Complementary DNA (cDNA) can be arranged and then immobilized on a substrate. The probes can be immobilized by covalent means such as by chemical bonding procedures or UV. In one such method, a cDNA is bound to a glass surface which has been modified to contain epoxide or aldehyde groups. In another case, a cDNA probe is placed on a polylysine coated surface and then UV cross-linked (Shalon et al. PCT publication WO95/35505, herein incorporated by reference). In yet another method, a DNA is actively transported from a solution to a given position on a substrate by electrical means (Heller et al. U.S. Pat. No. 5,605,662). Alternatively, individual DNA clones can be gridded on a filter. Cells are lysed, proteins and cellular components degraded and the DNA coupled to the filter by UV cross-linking.

[0068] Furthermore, the probes do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group. The linker groups are typically about 6 to 50 atoms long to provide exposure to the attached polynucleotide probe. Preferred linker groups include ethylene glycol oligomers, diamines, diacids and the like. Reactive groups on the substrate surface react with one of the terminal portions of the linker to bind the linker to the substrate. The other terminal portion of the linker is then functionalized for binding the polynucleotide probe.

[0069] The polynucleotide probes can be attached to a substrate by dispensing reagents for probe synthesis on the substrate surface or by dispensing preformed DNA fragments or clones on the substrate surface. Typical dispensers include a micropipette delivering solution to the substrate with a robotic system to control the position of the micropipette with respect to the substrate. There can be a multiplicity of dispensers so that reagents can be delivered to the reaction regions simultaneously.

[0070] Sample Preparation

[0071] In order to conduct sample analysis, a sample containing target polynucleotides is provided. The samples can be any sample containing target polynucleotides and obtained from any bodily fluid (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations.

[0072] DNA or RNA can be isolated from the sample according to any of a number of methods well known to those of skill in the art. For example, methods of purification of nucleic acids are described in Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed. Elsevier, New York, N.Y. (1993). In one case, total RNA is isolated using the TRIZOL total RNA isolation reagent (Life Technologies, Gaithersburg, Md.) and mRNA is isolated using oligo d(T) column chromatography or glass beads. Alternatively, when target polynucleotides are derived from an mRNA, the target polynucleotides can be a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from that cDNA, an RNA transcribed from the amplified DNA, and the like. When the target polynucleotide is derived from DNA, the target polynucleotide can be DNA amplified from DNA or RNA reverse transcribed from DNA. In yet another alternative, the targets are target polynucleotides prepared by more than one method.

[0073] When target polynucleotides are amplified it is desirable to amplify the nucleic acid sample and maintain the relative abundances of the original sample, including low abundance transcripts. Total mRNA can be amplified by reverse transcription using a reverse transcriptase and a primer consisting of oligo d(T) and a sequence encoding the phage T7 promoter to provide a single stranded DNA template. The second cDNA strand is polymerized using a DNA polymerase and a RNAse which assists in breaking up the DNA/RNA hybrid. After synthesis of the double stranded cDNA, T7 RNA polymerase can be added and RNA transcribed from the second cDNA strand template (Van Gelder et al. U.S. Pat. No. 5,545,522). RNA can be amplified in vitro, in situ or in vivo (See Eberwine, U.S. Pat. No. 5,514,545).

[0074] It is also advantageous to include quantitation controls within the sample to assure that amplification and labeling procedures do not change the true distribution of target polynucleotides in a sample. For this purpose, a sample is spiked with a known amount of a control target polynucleotide and the composition of polynucleotide probes includes reference polynucleotide probes which specifically hybridize with the control target polynucleotides. After hybridization and processing, the hybridization signals obtained should reflect accurately the amounts of control target polynucleotide added to the sample.

[0075] Prior to hybridization, it may be desirable to fragment the nucleic acid target polynucleotides. Fragmentation improves hybridization by minimizing secondary structure and cross-hybridization to other nucleic acid target polynucleotides in the sample or noncomplementary polynucleotide probes. Fragmentation can be performed by mechanical or chemical means.

[0076] The target polynucleotides may be labeled with one or more labeling moieties to allow for detection of hybridized probe/target polynucleotide complexes. The labeling moieties can include compositions that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means. The labeling moieties include radioisotopes, such as ³²p, ³³p or ³⁵S, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, such as fluorescent markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, and the like.

[0077] Exemplary dyes include quinoline dyes, triarylmethane dyes, phthaleins, azo dyes, cyanine dyes and the like. Preferably, fluorescent markers absorb light above about 300 nm, preferably above 400 nm, and usually emit light at wavelengths at least greater than 10 nm above the wavelength of the light absorbed. Specific preferred fluorescent markers include fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 available from Amersham Pharmacia Biotech (Piscataway, N.J.).

[0078] Labeling can be carried out during an amplification reaction, such as polymerase chain and in vitro transcription reactions, or by nick translation or 5′ or 3′-end-labeling reactions. In one case, labeled nucleotides are used in an in vitro transcription reaction. When the label is incorporated after or without an amplification step, the label is incorporated by using terminal transferase or by kinasing the 5′ end of the target polynucleotide and then incubating overnight with a labeled oligonucleotide in the presence of T4 RNA ligase.

[0079] Alternatively, the labeling moiety can be incorporated after hybridization once a probe/target complex has formed. In one case, biotin is first incorporated during an amplification step as described above. After the hybridization reaction, unbound nucleic acids are rinsed away so that the only biotin remaining bound to the substrate is that attached to target polynucleotides that are hybridized to the polynucleotide probes. Then, an avidin-conjugated fluorophore, such as avidin-phycoerythrin, that binds with high affinity to biotin is added. In another case, the labeling moiety is incorporated by intercalation into preformed target/polynucleotide probe complexes. In this case, an intercalating dye such as a psoralen-linked dye can be employed.

[0080] Under some circumstances it may be advantageous to immobilize the target polynucleotides on a substrate and have the polynucleotide probes bind to the immobilized target polynucleotides. In such cases the target polynucleotides can be attached to a substrate as described above.

[0081] Hybridization and Detection

[0082] Hybridization causes a denatured polynucleotide probe and a denatured complementary target to form a stable duplex through base pairing. Hybridization methods are well known to those skilled in the art (See, for example, Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen, ed. Elsevier, New York, N.Y. (1993)). Conditions can be selected for hybridization where exactly complementary target and polynucleotide probe can hybridize, i.e., each base pair must interact with its complementary base pair. Alternatively, conditions can be selected where target and polynucleotide probes have mismatches but are still able to hybridize. Suitable conditions can be selected, for example, by varying the concentrations of salt or formamide in the prehybridization, hybridization and wash solutions, or by varying the hybridization and wash temperatures.

[0083] Hybridization can be performed at low stringency with buffers, such as 6×SSPE with 0.005% Triton X-100 at 37° C., which permits hybridization between target and polynucleotide probes that contain some mismatches to form target polynucleotide/probe complexes. Subsequent washes are performed at higher stringency with buffers, such as 0.5×SSPE with 0.005% Triton X-100 at 50° C., to retain hybridization of only those target/probe complexes that contain exactly complementary sequences. Alternatively, hybridization can be performed with buffers, such as 5×SSC/0.2% SDS at 60° C. and washes are performed in 2×SSC/0.2% SDS and then in 0.1×SSC. Stringency can also be increased by adding agents such as formamide. Background signals can be reduced by the use of detergent, such as sodium dodecyl sulfate, Sarcosyl or Triton X-100, or a blocking agent, such as sperm DNA.

[0084] Hybridization specificity can be evaluated by comparing the hybridization of specificity-control polynucleotide probes to specificity-control target polynucleotides that are added to a sample in a known amount. The specificity-control target polynucleotides may have one or more sequence mismatches compared with the corresponding polynucleotide probes. In this manner, whether only complementary target polynucleotides are hybridizing to the polynucleotide probes or whether mismatched hybrid duplexes are forming is determined.

[0085] Hybridization reactions can be performed in absolute or differential hybridization formats. In the absolute hybridization format, target polynucleotides from one sample are hybridized to the probes in a microarray format and signals detected after hybridization complex formation correlate to target polynucleotide levels in a sample. In the differential hybridization format, the differential expression of a set of genes in two biological samples is analyzed. For differential hybridization, target polynucleotides from both biological samples are prepared and labeled with different labeling moieties. A mixture of the two labeled target polynucleotides is added to a microarray. The microarray is then examined under conditions in which the emissions from the two different labels are individually detectable. Probes in the microarray that are hybridized to substantially equal numbers of target polynucleotides derived from both biological samples give a distinct combined fluorescence (Shalon et al. PCT publication WO95/35505). In a preferred embodiment, the labels are fluorescent labels with distinguish-able emission spectra, such as a lissamine conjugated nucleotide analog and a fluorescein conjugated nucleotide-analog. In another embodiment Cy3/Cy5 fluorophores (Amersham Pharmacia Biotech) are employed.

[0086] After hybridization, the microarray is washed to remove nonhybridized nucleic acids and complex formation between the hybridizable array elements and the target polynucleotides is detected.

[0087] Methods for detecting complex formation are well known to those skilled in the art. In a preferred embodiment, the target polynucleotides are labeled with a fluorescent label and measurement of levels and patterns of fluorescence indicative of complex formation is accomplished by fluorescence microscopy, preferably confocal fluorescence microscopy. An argon ion laser excites the fluorescent label, emissions are directed to a photomultiplier and the amount of emitted light detected and quantitated. The detected signal should be proportional to the amount of probe/target polynucleotide complex at each position of the microarray. The fluorescence microscope can be associated with a computer-driven scanner device to generate a quantitative two-dimensional image of hybridization intensity. The scanned image is examined to determine the abundance/expression level of each hybridized target polynucleotide.

[0088] In a differential hybridization experiment, target polynucleotides from two or more different biological samples are labeled with two or more different fluorescent labels with different emission wavelengths. Fluorescent signals are detected separately with different photomultipliers set to detect specific wavelengths. The relative abundances/expression levels of the target polynucleotides in two or more samples is obtained.

[0089] Typically, microarray fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one microarray is used under similar test conditions. In a preferred embodiment, individual polynucleotide probe/target complex hybridization intensities are normalized using the intensities derived from internal normalization controls contained on each microarray.

[0090] Expression Profiles

[0091] This section describes an expression profile using the composition of this invention. The expression profile can be used to detect changes in the expression of genes implicated in blood cell biology. These genes include genes whose altered expression is correlated with cancer, immunopathology, apoptosis and the like.

[0092] The expression profile comprises the polynucleotide probes of the invention. The expression profile also includes a plurality of detectable complexes. Each complex is formed by hybridization of one or more polynucleotide probes to one or more complementary target polynucleotides. At least one of the polynucleotide probes, preferably a plurality of polynucleotide probes, is hybridized to a complementary target polynucleotide forming, at least one, preferably a plurality of complexes. A complex is detected by incorporating at least one labeling moiety in the complex. The labeling moiety has been described above.

[0093] The expression profiles provide “snapshots” that can show unique expression patterns that are characteristic of a disease or condition.

[0094] Utility of the Invention

[0095] The composition comprising a plurality of polynucleotide probes can be used as hybridizable elements in a microarray. Such a microarray can be employed in several applications including diagnostics, prognostics and treatment regimens, drug discovery and development, toxicological and carcinogenicity studies, forensics, pharmacogenomics and the like.

[0096] In one situation, the microarray is used to monitor the progression of disease. Researchers can assess and catalog the differences in gene expression between healthy and diseased tissues or cells. By analyzing changes in patterns of gene expression, disease can be diagnosed at earlier stages before the patient is symptomatic.

[0097] Similarly, the invention can be used to monitor the progression of disease or the efficacy of treatment. For some treatments with known side effects, the microarray is employed to “fine tune” the treatment regimen. A dosage is established that causes a change in genetic expression patterns indicative of successful treatment. Expression patterns associated with undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or manifest symptoms, before altering the course of treatment.

[0098] Alternatively, animal models which mimic a disease, rather than patients, can be used to characterize expression profiles associated with a particular disease or condition. For example, a characteristic gene expression pattern for the graft versus host reaction can be generated using analogous reactions that occur when lymphocytes from one donor are mixed with lymphocytes from another donor. This gene expression data may be useful in diagnosing and monitoring the course of graft versus host reaction in a patient, in determining gene targets for intervention, and in testing novel immunosuppressants.

[0099] The composition is particularly useful for diagnosing and monitoring the progression of diseases that are associated with the altered expression of genes implicated in blood cell biology. The expression of these genes is associated with cellular processes such as hematopoiesis, immunological responses, immunopathology, cell proliferation, apoptosis, and the like. Thus, the microarray is particularly useful to diagnose immunopathologies including, but not limited to, AIDS, Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitus, Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritable bowel syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, and autoimmune thyroiditis;viral, bacterial, fungal, parasitic, and protozoal infections and trauma.

[0100] The invention also allows researchers to develop sophisticated profiles of the effects of currently available therapeutic drugs. Tissues or cells treated with these drugs can be analyzed and compared to untreated samples of the same tissues or cells. In this way, an expression profile of known therapeutic agents will be developed. Knowing the identity of sequences that are differentially regulated in the presence and absence of a drug will allow researchers to elucidate the molecular mechanisms of action of that drug.

[0101] Also, researchers can use the microarray to rapidly screen large numbers of candidate drugs, looking for ones that have an expression profile similar to those of known therapeutic drugs, with the expectation that molecules with the same expression profile will likely have similar therapeutic effects. Thus, the invention provides the means to determine the molecular mode of action of a drug.

[0102] It is understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. The examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention.

[0103] For purposes of example, the preparation and sequencing of the LNODNOT03 cDNA library, from which Incyte Clones 1573272, 1573553, 1574415, 1574617, 1574637, and 1576661 were isolated, is described in detail. Preparation and sequencing of cDNA libraries in the LifeSeq® database (Incyte) have varied over time, and the gradual changes involved use of kits, plasmids, and machinery available at the particular time the library was made and analyzed.

[0104] The LNODNOT03 cDNA library was constructed from microscopically normal lymph node tissue excised from a 67-year-old Caucasian male. This tissue was associated with tumorous lung tissue. The patient history included squamous cell carcinoma of the lower lobe, benign hypertension, arteriosclerotic vascular disease, and tobacco abuse. The patient was taking Doxycycline, a tetracycline, to treat an infection.

[0105] The frozen tissue was homogenized and lysed using a POLYTRON homogenizen PT-3000 (Brinkmann Instruments, Westbury, N.J.) in guanidinium isothiocyanate solution. The lysate was centrifuged over a 5.7 M CsCl cushion using an SW28 rotor in a L8-70M Ultracentrifuge (Beckman Coulter Fullerton, Calif.) for 18 hours at 25,000 rpm at ambient temperature. The RNA was extracted with acid phenol pH 4.7, precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in RNAse-free water, and DNase treated at 37° C. The RNA extraction was repeated with acid phenol pH 4.7 and precipitated with sodium-acetate and ethanol as before. The mRNA was then isolated using the OLIGO.TEX kit (QIAGEN, Inc., Chatsworth, Calif.) and used to construct the cDNA library.

[0106] The mRNA was handled according to the recommended protocols in the SUPERSCRIPT Plasmid, System for cDNA Synthesis and Plasmid Cloning (Life Technologies). cDNAs were fractionated on SEPHAROSE CL4B column (Amersham Pharmacia Biotech), and those cDNAs exceeding 400 BP were ligated into pSPORT I. The plamid PSORT I plasmid (Life Technologies) was subsequently transformed into DH5a competent cells (Life Technologies).

[0107] In some cases, cDNA libraries have been normalized in a single round according to the procedure of Soares et al. ((1994), Proc. Natl. Acad. Sci. USA91: 9928-9932) with the following modifications. The primer to template ratio in the primer extension reaction was increased from 2:1 to 10:1. The dNTP concentration in the reaction was reduced to 150 μM each dNTP, allowing the generation of longer (400-1000 nt) primer extension products. The reannealing hybridization was extended from 13 to 19 hours. The single stranded DNA circles of the normalized library were purified by hydroxyapatite chromatography and converted to partially double-stranded by random priming, followed by electroporation into DH10B competent bacteria (Life Technologies).

[0108] The Soares normalization procedure is designed to reduce the initial variation in individual cDNA frequencies to achieve abundances within one order of magnitude while maintaining the overall sequence complexity of the library. In the normalization process, the prevalence of high-abundance cDNA clones decreases significantly, clones with mid-level abundance are relatively unaffected, and clones for rare transcripts are effectively increased in abundance. In the modified Soares normalization procedure, significantly longer hybridization times are used which allows for the increase of gene discovery rates by biasing the normalized libraries toward low-abundance cDNAs that are well represented in a standard transcript image.

[0109] Plasmid cDNA was released from the cells and purified using the REAL Prep 96 plasmid kit (Catalog #26173, QIAGEN). The recommended protocol was employed except for the following changes: 1), the bacteria were cultured in 1 ml of sterile Terrific Broth (Life Technologies) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures were incubated for 19 hours and at the end of incubation, the cells were lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the last step in the protocol, samples were transferred to a 96-well block for storage at 4° C.

[0110] cDNAs were sequenced according to the method of Sanger et al. ((1975) J. Mol. Biol. 94: 441f), using the Perkin Elmer Catalyst 800 or a MICROLAB 2200 (Hamilton, Reno, Nev.) in combination with DNA ENGINE Thermal Cyclers (PTC200 from MJ Research, Watertown, Mass.) and ABI PRISM 377 on 373 DNA Sequencing Systems (PE Biosystems, Foster City, Calif.) and the reading frame was determined.

[0111] As used herein, “homology” refers to sequence similarity between a reference sequence and at least a portion of a newly sequenced clone insert, and can refer to either a nucleic acid or amino acid sequence. The Genbank databases which contain previously identified and annotated sequences, were searched for regions of homology using BLAST (Basic Local Alignment Search Tool). (See, e.g., Altschul, S. F. (1993) J. Mol. Evol. 36: 290-300; and Altschul et al. (1990) J. Mol. Biol. 215: 403-410.)

[0112] BLAST involves first finding similar segments between the query sequence and a database sequence, then evaluating the statistical significance of any matches that are found and finally reporting only those matches that satisfy a user-selectable threshold of significance. BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. The fundamental unit of the BLAST algorithm output is the High scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary, but equal lengths, whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user.

[0113] The basis of the search is the product score, which is defined as: $\frac{\%\mathrm{sequence}\mathrm{identit}y\times \%\mathrm{maximum}\mathrm{BLAST}\mathrm{score}}{100}$

[0114] The product score takes into account both the degree of similarity (identity) between two sequences and the length of the sequence match as reflected in the BLAST score. The BLAST score is calculated by scoring +5 for every base that matches in an HSP and −4 for every mismatch. For example, with a product score of 40, the match will be exact within a 1% to 2% error, and, with a product score of 70, the match will be exact. Homologous molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules. The P-value for any given HSP is a function of its expected frequency of occurrence and the number of HSPs observed against the same database sequence with scores at least as high.

[0115] Transcript profiles were generated using the Lifeseq® Transcript Imaging tool (Incyte). To identify genes that are differentially expressed in hematopoietic or inflamed biological samples, reverse transcript profiles from specific target cDNA library pools derived from either hematopoietic or inflamed biological samples were obtained. The number of cDNA libraries in a cDNA library pool varied from one cDNA library member to 6 cDNA library members. For library pools which contained more than one member, an averaged reverse transcript image was obtained. The target library pools were the following: 1. ADENINB01; 2. COLSUCT01, COLNNOT23, SINTNOT13; 3. COLNCRT01, COLNNOT27, SINTBST01; 4. ENDCNOT01, ENDCNOT02, ENDCNOT03, HUVELPB01, HUVENOB01, HUVESTB01; 5. EOSIHET02, UCMCL5T01; 6. HMC1NOT01; 7. LNODNOT02, LNODNOT03; 8. LUNGAST01; 9. MMLR1DT01, MMLR2DT01, MMLR3DT01; 10. NEUTFMT01, NEUTGMT01, NEUTLPT01; 11. PANCDIT01, 12. TONSNOT01; 13. SYNOOAT01; 14. SYNORAB01, SYNORAT01, SYNORAT03, SYNORAT04, SYNORAT05; 15. THP1PLB01, THP1PLB02; and 16. TMLR2DT01, TMLR3DT01, TMLR3DT02.

[0116] Reverse transcript profiles were also derived from 39 subtraction cDNA libraries which were derived from predominantly nonhematopoetic noninflamed biological samples. The following is a list of the cDNA libraries: FIBRAGT01, FIBRAGT02, FIBRANT01, FIBRNGT01, FIBRNGT02, FIBRSEM01, KERANOT01, COLNNOM01, COLNTUM01, EYECNOM01, FIBRFEM01, HNT2NOM01, OVARTUM02, PANCTUM01, UTRPNOM01, CARDFEM01, FIBRSEM01, PANCISM01, LUNGFEM01, PTHYTUM01, BRAINOM01, BRAINOM02, BRAINOM03, BRSTNOM01, BRSTNOM02, COCHFEM01, LIVRNOM01, LUNGNOM01, MELANOM01, NERVMSM01, OLFENOM01, OVARNOM01, OVARTUM01, PINENOM01, PLACNOM01, PLACNOM02, PLACNOM03, RETNNOM01, and RETNNOM02. In some cases, one or more subtraction libraries were derived from hematopoietic or inflamed biological samples, such as the NERVMSM01 library derived from the tissue of a patient suffering from multiple sclerosis. An averaged subtraction transcript profile was obtained by poling the transcript information from all 39 libraries.

[0117] The LifeSeq® Multiple Transcript Subsetting tool (Incyte) was used to subtract the averaged subtraction transcript profile from each target cDNA library pool. A list of subtracted transcripts which consisted of clustered ESTs was generated. The subtracted transcripts were ranked according to abundance. The 40 most abundant transcripts were selected from each set of subtracted library pools.

[0118] To identify additional genes that are differentially expressed in immunological responses, reverse transcript profiles from specific target cDNA libraries derived from inflamed biological samples were obtained. Subtraction cDNA library transcript profiles were generated from healthy counterparts. In some cases the subtraction transcript profile was created by averaging transcript images from a pool of cDNA libraries. Target and subtraction cDNA libraries are listed in the following table.

[0119] A list of subtracted transcripts which consisted of clustered ESTs was generated. After subtracting transcript profiles, all upregulated sequences were selected.

[0120] To identify novel genes that are abundantly expressed in an immunological response, 43 cDNA libraries derived from hematopoietic or inflamed biological samples were picked. These libraries were: ADENINB01, AMLBNOT01, BMARNOR02, BMARNOT02, BMARNOT03, EOSIHET02, HMC1NOTO01, LEUKNOT02, LEUKNOT03, LNODNOT02, LNODNOT03, MMLR1DT01, MMLR2DT01, MMLR3DT01, MPHGLPT02, MPHGNOT02, MPHGNOT03, NEUTFMT01, NEUTGMT01, NEUTLPT01, THYMNOT02, TLYMNOT01, TLYMNOT02, TMLR2DT01, TMLR3DT01, TMLR3DT02, SPLNFET01, SPLNFET02, SPLNNOT02, SPLNNOT04, TBLYNOT01, THP1NOB01, THP1NOT01, THP1NOT03, THP1AZT01, THP1PEB01, THP1PLB01, THP1PLB02, THP1T7T01, TONSNOT01, U937NOT01, UCMCL5T01, and UCMCNOT02.

[0121] Reverse transcript profiles were generated from the 43 cDNA libraries using a product score of 100 as the maximum cutoff. Using this setting returns all the sequences in the transcript profile. The top most abundant sequences were selected. Reverse transcript profiles were also generated from the chosen libraries using a product score of 70 as the maximum cutoff. This procedure effectively removed exact matches to gene sequences found in GenBank. A list of transcripts which consisted of clustered ESTs was generated. The top 20 most abundant sequences were selected.

[0122] To identify genes known to be associated with the regulation of blood cell biology, the literature was surveyed and relevant sequences in GenBank and Lifeseq® databases were identified. Genes were selected on the following basis. Genes were identified from the literature that are involved in blood cell biology. Then GenBank database and Lifeseq® databases (Incyte) were screened to identify other genes containing homologous sequences using BLAST. Additionally, the Lifeseq® database (Incyte) was searched using a group of key words including cyclin, cul, phosphatase, apoptosis, kinase, serine kinase, tyrosine kinase, cdk, phosphodiesterase, protease, protease inhibitor, metalloproteinase, cathepsin, phospholipase, E2F, integrin, receptor, cytochrome, p450, cox, lipocortin, retinoic acid, CD, cdc, fas, TNF, gadd, cytokine, chemokine, growth factor, interleukin, heat shock protein, HSP, stress, STAT, myb, jun, fos, dpl, myc, bak, bcl, p53, phox, inflammation, oxidase, and glutathione.

[0123] After selecting the transcripts (ESTs) of interest, partial sets of ESTs representing a single gene were identified. Partial sets of ESTs for the same gene but identified by different selection methods were clustered. Sets of partial sequences (and their quality scores) were used to assemble contiguous sequences using the Phrap program with default settings. The longest “high quality” set of partial sequences was chosen. “High quality” is defined as sequences starting and ending with at least 10 contiguous base calls with quality scores above 12. When performing the clustering process, the full Lifeseq® database (Incyte) was searched in order to obtain the longest “high quality” set of partial sequences.

[0124] After clustering related ESTs, EST clusters were checked for redundancy. The cDNAs were compared to each other using the BLASTn database search program. Any two sequences with similarity scores greater than 250 and percent identities greater than 95% were considered redundant. A representative cDNA sequence from each redundancy set was chosen and corresponds to the EST with the longest read sequence. In some cases, the representative cDNA sequence did not originate from the original cDNA libraries used in the selection processes.

[0125] Full length cDNAs (identified from Genbank or database and database (Incyte) Lifeseq®) and EST sets were also compared with each other to remove redundant sequences.

[0126] Illustrative polynucleotide probes for use in this invention are provided in the Sequence Listing and are SEQ ID Nos: 1-1508. The polynucleotide probes are derived from genes implicated in blood cell biology, including hematopoiesis, immunological responses and immuno-pathology. Of the 1,508 nonredundant polynucleotide probes 43% were exact matches to sequences in the public domain, 57% were homolgous to public domain sequences or unique sequences which are abundantly or differentially expressed in blood cell biology. Some of the public domain sequences were not known to be abundantly expressed or differentially expressed in hematopoiesis or immunological responses.

[0127] A microarray was prepared as follows: 96 different PCR polynucleotide probes were laid down in quadruplicate (4 arrays 100 spots each) on a aldehyde derivatized slide available from Cel Associates (Houston, Tex.). The glass slide had dimensions of 18×24 mm. The distance between the spots on the array is 500 microns. Samples were printed on the glass slide from the left upper corner to the right lower corner in the following order: g177865 (Human tumor necrosis factor alpha); g177869 (Human alpha-2-macroglobulin); g178163 (Human ADP-ribosylation factor 1); g219475 (Human immediate-early-response); g179699 (Human C5a anaphylatoxin receptor); g179892 (Human CAMP phosphodiesterase); g184840 (Human Fc-gamma receptor I); g181181 (Human cathepsin G); g181485 (Human DNA-binding protein B); g182487 (Human Fc-epsilon-receptor gamma-chain); g182504 (Human ferritin H chain mRNA); g182632 (Human FKBP-12 protein); g182976 (Human glyceraldehyde-3-phosphate dehydrogenase); g183063 (Human glia-derived nexin); g183067 (Human mRNA sequence with homology to GDP binding protein); g31914 (Human mRNA for coupling protein G(s) alpha); g184420 (Human 90-kDa heat-shock protein); g186264 (Human gamma-interferon-inducible protein); g179579 (Human beta-thromboglobulin-like protein); g187172 (Human leukotriene A-4 hydrolase); g187220 (Human L-plastin gene); g187243 (Human lysozyme mRNA); g188255 (Human MHC class II HLA-DR-alpha); g189150 (Human nephropontin); g190813 (Human Wilm's tumor-related protein); g189267 (Human neutrophil oxidase factor; g219868 (Human HM89); g182482 (Human fibroblast collagenase inhibitor); g189546 (Human plasminogen activator inhibitor); g899458 (Human 14-3-3 protein); g184628 (Human interleukin 6); g250802 (cathepsin S); g264772 (thymosin beta-10); g28251 (beta-actin.); g291926 (Human cystatin B); g292416 (Human macrophage inflammatory protein); g29508 (Human BTG1); g181179 (Human cathepsin D); g179952 (Human cathepsin L); g186933 (Human leukocyte adhesion protein); g29793 (leukocyte antigen CD37); g184628 (Human interleukin 6);

[0128] g29850 (Human CDw40 nerve growth factor); g238776 (p55); g306467 (Human binding protein mRNA); g306486 (Human cap-binding protein); g306773 (Human GM-CSF receptor); g307165 (Human myeloid cell differentiation protein); g307374 (Human RHOA proto-oncogene multi-drug-resistance); g31097 (Human mRNA for elongation factor 1 alpha); g32576 (Human mRNA for interleukin-1 receptor); g220063 (Human sphingolipid activator proteins); g186283 (Human interleukin 1-beta); g190419 (Human secretory granule proteoglycan peptide); g33917 (Human mRNA for gamma-interferon inducible protein); g339420 (Human T cell-specific protein (RANTES)); g339688 (Human thymosin beta-4 mRNA); g339690 (Human prothymosin alpha); g339737 (Human tumor necrosis factor (TNF)); g340020 (Human alpha-tubulin); g34312 (Human mRNA for lactate dehydrogenase-A); g187434 (Human monocyte chemotactic and activatinG factor); g179579 (Human beta-thromboglobulin-like protein); g34625 (Human gene for melanoma growth stimulator); g188558 (Human macrophage inflam-matory protein); g181191 (Human cathepsin B proteinase); g348911 (Human glycoprotein); g337494 (Human ribosomal protein L7a); g35517 (Human mRNA for pleckstrin (P47)); g1562497 (Human poly(A)-binding protein); g338285 (Human manganese-containing superoxide dismutase); g339737(Human tumor necrosis factor (TNF)); g404012 (Human pre-B cell enhancing factor); g495286 (Human melanoma differentiation associated factor); g416368 (Human CTLA4 counter-receptor); g433415 (Human mRNA for DNA-binding protein, TAXR); g434760 (Human mRNA for ORF); g598867 (Human HepG2 partial cDNA); g450280 (Human sui1iso1); g517197 (Human urokinase-type plasminogen activator); g187151 (Human lysosomal acid lipase); g468150 (Human MAP kinase); g496975 (Human cyclooxygenase-2); g186512 (Human (clone 1950.2) interferon-gamma); g560790 (Human mRNA for calgizzarin); g1255239 (Human lysosomal-associated multitransmembrane protein); g245388 (beta 2-microglobulin); g178019 (Human cytokine (SCYA2) gene); g1304482 (Human tissue inhibitor of metalloprotein); g886049 (Human Ich-2 cysteine protease); g189177 (Human nuclear factor kappa-B DNA binding protein); g37983 (Human mRNA of X-CGD gene); g178083 (Human adenylyl cyclase-associated protein); g29899 Human mRNA for c-fms proto-oncogene); and g36606 (Human spermidine/spermine N1-acetyltransferase). The 9th and the 18th rows contained controls derived from Drosophila melonogasterand Arabidopsis thaliana.

[0129] An experiment was performed to measure gene expression in A. THP1 cells and B. THP1 cells first treated with 100 ng/ml PMA (phorbol ester myristate) for 48 hours and then treated with 1 microgram/ml LPS (liposacharride) for 48 hours. THP1 cells were obtained for the American Type Culture Collection. Treated and untreated cells were lysed, mRNA was purified using oligo d(T) chromatography. The mRNA was labeled by adding oligo(dT)20 (5 micrograms) to 2 micrograms mRNA and heating the reaction to 70° C. Hybridization was performed with 5×SSC/0.2% SDS for six (6) hours at 60° C. The slides were washed for five (5) minutes in 1×SSC/0.2%SDS at 30° C. followed by five (5) minutes in 0.1×SSC/0.2 SDS at room temperature followed by 30 minutes in 0.1×SCC at room temperature. Signals were detected using a custom made confocal fluorescence microscope.

[0130] Gene expression analysis was performed to identify gene sequences which were expressed at higher levels in treated THP1 cells rather than untreated THP1 cells. FIG. 1 shows the gene expression pattern observed in untreated THP1 cells. FIG. 2 shows the gene expression pattern observed in treated THP1 cells. By a comparision of both expression patterns, those genes differentially expressed in treated THP1 cells can be identified. For example, genes that are highly expressed in treated THP1 cells include the macrophage inflammatory protein gene, the cytokine (SCYA2) gene, and beta-thromboglobulin gene among others.

[0131] ADENINB01 Library was constructed using RNA isolated from the inflamed adenoid tissue of a 3-year-old child. (RNA came from Clontech.) cDNA synthesis was initiated using a combination of oligo(dT) and random priming. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0132] BMARNOT02 Library was constructed using RNA isolated from the normal bone marrow of 24 male and female Caucasian donors, 16 to 70 years old.(RNA came from Clontech.) cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Iambda UniZAP vector.

[0133] BMARNOT02 Library was constructed using RNA isolated from the normal bone marrow of 24 male and female Caucasian donors, 16 to 70 years old. (RNA came from Clontech.) cDNA synthesis was initiated using a random primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0134] BMARNOT03 Library was constructed using 1 microgram of polyA RNA isolated from the nontumorous left tibial bone marrow tissue of a 16-year-old Caucasian male during a partial left tibial ostectomy with free skin graft. Pathology indicated the distal bone marrow and soft tissue margins were negative for tumor. Pathology for the associated tumor tissue, situated within the proximal 7 cm of the left tibia, indicated metastatic alveolar rhabdomyo sarcoma. The tumor perforated the cortical bone anteriorly into the soft tissue, with extension into the articular space. The patient presented with lower leg joint pain. Patient history included an abnormality of the red blood cells. Previous surgeries included bone and bone marrow biopsy, and soft tissue excision. The patient was treated with radiation to the buttock (8 weeks total in 1994, 1995). Patient medications included Vincristine, Adriamycin, and Cytoxan. Family history included osteoarthritis in the mother and alcohol abuse in the father. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0135] COLNCRT01 Library was constructed using 0.99 micrograms of polyA RNA isolated from a diseased section of the ascending colon of a 40- year-old Caucasian male during a partial colectomy. Pathology indicated Crohn's disease involving the proximal colon and including the cecum. The ascending and transverse colon displayed linear ulcerations and skip lesions. There was transmural inflammation but no fistulas. The ileum was uninvolved. There was also a benign carcinoid tumor in the tip of the appendix. Patient history included anorexia nervosa, candidiasis of the mouth, Type I diabetes, diarrhea, viral meningitis, polyp of the vocal cord, and tobacco use. Previous surgeries included repair of an inguinal hernia. Patient medications included Zantac (ranitidine), Prednisone, Annusol suppositories, and insulin. Family history included hypertension in the mother. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0136] COLNNOT05 Library was constructed using 1.4 micrograms of polyA RNA isolated from the normal sigmoid colon tissue of a 40-year-old Caucasian male during a partial colectomy. Pathology indicated Crohn's disease involving the proximal colon and including the cecum. The ascending and transverse colon displayed linear ulcerations and skip lesions. There was transmural inflammation but no fistulas. The ileum was uninvolved. There was also a benign carcinoid tumor in the tip of the appendix. Patient history included anorexia nervosa, candidiasis of the mouth, Type I diabetes, diarrhea, viral meningitis, polyp of the vocal cord, and tobacco use. Previous surgeries included repair of an inguinal hernia. Patient medications included Zantac (ranitidine), Prednisone, Annusol suppositories, and insulin. Family history included hypertension in the mother. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector. COLNCRT01 is a diseased ascending colon library from the same donor.

[0137] COLNNOT23 Library was constructed using 1 microgram of polyA RNA isolated from diseased colon tissue removed from a 16-year-old Caucasian male during a total colectomy with abdominal/perineal resection. Pathology indicated gastritis and pancolonitis consistent with the acute phase of ulcerative colitis. (The process is characterized by acute colitis with crypt abscess formation.) Inflammation was more severe in the transverse colon, with inflammation confined to the mucosa. There was only mild involvement of the ascending and sigmoid colon, and no significant involvement of the cecum, rectum, or terminal ileum. The patient presented with blood in the stool, anemia, abdominal pain, nausea, and vomiting. Patient medications included Minocin, Cephaxelin, Prednisone, Flagyl, Zantac (ranitidine), cortisone enemas, omrprazole, iron, dextran, and cyclosporin. Family history included irritable bowel syndrome, hypertension, and atherosclerotic coronary artery disease in a grandparent. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0138] COLNNOT27 Library was constructed using 1 microgram of polyA RNA isolated from diseased cecal tissue removed from 31-year-old Caucasian male during a total intra-abdominal colectomy, appendectomy, and permanent ileostomy. Pathology indicated severe active Crohn's disease involving the colon from the cecum to the rectum. There were deep rake-like ulcerations which spared the intervening mucosa. The ulcers extended into the muscularis, and there was transmural inflammation. The ileum and appendix were uninvolved. The patient presented with enteritis and diarrhea. Patient history included an irritable colon. Previous surgeries included a colonscopy. Patient medications included Asacol (mesalamine), Flagyl (metronidazole), Azulfidine (sulfasalazine), and Prednisone (glucocorticoid). cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to Acari adaptors, digested with Not, size-selected, and cloned into the Not and Acari sites of the pINCY vector (Incyte).

[0139] COLSUCT01 Library was constructed using 1 microgram of polyA RNA isolated from diseased sigmoid colon tissue removed from a 70-year-old Caucasian male during colectomy with permanent ileostomy. Pathology indicated chronic ulcerative colitis in the distal 25 cm of the colon with acute and chronic inflammation and architectural distortion in the area. Chronic ulcerative colitis was identified in the rectum and sigmoid colon. There was hyperplastic polyp in the ascending colon. The remaining colon, terminal ileum, appendix, and anus showed no diagnostic abnormality. The patient presented with functional diarrhea and blood in the stool. Patient history included benign neoplasm of the colon, hyperlipidemia, benign hypertension, atrial fibrillation, and tobacco use. Patient medications included Asacol (mesalamine) for colitis, Prednisone (glucocorticoid), Coumadine, Lanoxin, Hygroton, Zestril, and Rowasa. Family history included atherosclerotic coronary artery disease and a myocardial infarction in the father, atherosclerotic coronary artery disease and a myocardial infarction in the mother, atherosclerotic coronary artery disease and a myocardial infarction in the sibling(s), and atherosclerotic coronary artery disease in the grandparent(s). cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0140] ENDCNOT01 Library was constructed using 1 microgram of polyA RNA isolated from endothelial cells removed from the coronary artery of a 58-year-old Hispanic male. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0141] ENDCNOT02 Library was constructed using 1 microgram of polyA RNA isolated from dermal microvascular endothelial cells removed from a 30-year-old Caucasian female. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0142] ENDCNOT03 Library was constructed using 1 microgram of polyA RNA isolated from dermal microvascular endothelial cells removed from a neonatal Caucasian male. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0143] EOSIHET02 Library was constructed using RNA isolated from peripheral blood cells apheresed from a 48-year-old Caucasian male. Patient history included hypereosinophilia. Patient medications included hydroxyurea, allopruinol, warfarin, prednisone, and interferon alpha, ascorbic acid, and aspirin. The cell population was determined to be greater than 77% eosinophils by Wright's staining. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0144] HMC1NOT01 Library was constructed using RNA isolated from the HMC-1 human mast cell line derived from a 52-year-old female. Patient history included mast cell leukemia. Family history included atherosclerotic coronary artery disease and a joint disorder involving multiple joints in the mother; and cerebrovascular disease, diabetes insipidus, and tobacco abuse in the father. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0145] HUVENOB01 Library was constructed using RNA isolated from unstimulated HUV-EC-C (ATCC CRL 1730) cells. HUV-EC-C is an endothelial cell line derived from the vein of a normal human umbilical cord (ref: PNAS81:6413). RNA was made by lysing 2×10e 8 cells in GuSCN, followed by DNAse treatment. cDNA synthesis was initiated using a combination of oligo(dT) and random priming. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0146] HUVELPB01 Library was constructed using RNA isolated from HUV-EC-C (ATCC CRL 1730) cells that were stimulated with cytokine/LPS. HUV-EC-C is an endothelial cell line derived from the vein of a normal human umbilical cord (ref: PNAS81:6413). RNA was isolated from two pools of HUV-EC-C cells that had been treated with either gamma IFN and TNF-alpha or IL-1 beta and LPS. In the first instance, HUV-EC-C cells were treated with 4 units/ml TNF and 2 units/ml IFNg for 96 hours at a density of 4.9×10e8 cells/ml. The yield was 1296 micrograms of total RNA, from which 11 microgrammes of polyA was obtained (0.8% recovery). In the second instance, cells were treated with 1 units/ml IL-1 and 100 ng/ml LPS for 5 hours. Density was 1×108 cells/ml. The yield was 1000 micrograms of RNA, from which 5.3 micrograms of polyA was isolated (0.5% recovery). cDNA synthesis was initiated using a combination of oligo(dT) and random priming. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0147] HUVELPB01 Library was constructed using RNA isolated from HUV-EC-C (ATCC CRL 1730) cells that were stimulated with cytokine/LPS. HUV-EC-C is an endothelial cell line derived from the vein of a normal human umbilical cord (ref: PNAS81:6413). RNA was isolated from two pools of HUV-EC-C cells that had been treated with either gamma IFN and TNF-alpha or IL-1 beta and LPS. In the first instance, HUV-EC-C cells were treated with 4 units/ml TNF and 2 units/ml IFNg for 96 hours at a density of 4.9×10e8 cells/ml. The yield was 1296 micrograms of total RNA, from which 11 micrograms of polyA was obtained (0.8% recovery). In the second instance, cells were treated with 1 units/ml IL-1 and 100 ng/ml LPS for 5 hours. Density was 1×108 cells/ml. The yield was 1000 micrograms of RNA, from which 5.3 micrograms of polyA was isolated (0.5% recovery). cDNA synthesis was initiated using a combination of oligo(dT) and random priming. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0148] HUVENOB01 Library was constructed using RNA isolated from unstimulated HUV-EC-C (ATCC CRL 1730) cells. HUV-EC-C is an endothelial cell line derived from the vein of a normal human umbilical cord (ref: PNAS81:6413). RNA was made by lysing 2×10e8 cells in GuSCN, followed by DNAse treatment. cDNA synthesis was initiated using a combination of oligo(dT) and random priming. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0149] HUVESTB01 Library was constructed using RNA isolated from shear-stressed HUV-EC-C (ATCC CRL 1730) cells. HUV-EC-C is an endothelial cell line derived from the vein of a normal human umbilical cord (ref: PNAS81:6413). Before RNA isolation, the cells were subjected to a shear stress of 10 dynes/cm. cDNA synthesis was initiated using a combination of oligo(dT) and random priming. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0150] LEUKNOT02 Library was constructed using 1 microgram of polyA RNA isolated from white blood cells of a 45-year-old female with blood type O+. The donor tested positive for cytomegalovirus (CMV). cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0151] LEUKNOT03 Library was constructed using 1 microgram of polyA RNA isolated from white blood cells of a 27-year-old female with blood type A+. The donor tested negative for cytomegalovirus (CMV). cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0152] LNODNOT02 Library was constructed using 1 microgram of polyA RNA isolated from the lymph node tissue of a 42-year-old Caucasian female, who died of cardiac arrest. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0153] LNODNOT03 Library was constructed using 1 microgram of polyA RNA isolated from lymph node tissue removed from a 67-year-old Caucasian male during a segmental lung resection and bronchoscopy. On microscopic exam, this tissue was found to be extensively necrotic with 10% viable tumor. Pathology for the associated tumor tissue indicated invasive grade 3-4 squamous cell carcinoma, forming a mass in the right lower lobe, which grossly puckers the pleura. Microscopically, tumor invaded into but not through the visceral pleura. Focally, tumor was seen obliterating the bronchial lumen. The bronchial margin was negative for dysplasia/neoplasm. One of two intrapulmonary lymph nodes was metastatically involved. One of four inferior mediastinal(subcarinal) and two of eight superior mediastinal (right lower paratracheal) lymph nodes were metastatically involved. Multiple lymph nodes were negative for tumor. A small component of grade 3 adenocarcinoma was present in the tumor, which manifested itself most prominently in some of the metastases in the regional lymph nodes. The patient presented with a cough. Patient history included hemangioma and tobacco abuse. Previous surgeries included appendectomy. Patient medications included doxycycline. Family history included atherosclerotic coronary artery disease, benign hypertension, and congestive heart failure in the mother; atherosclerotic coronary artery disease and congestive heart failure in the father; and atherosclerotic coronary artery disease in the grandparent(s). cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0154] LUNGAST01 Library was constructed using 2 micrograms of polyA RNA isolated from the lung tissue of a 17-year-old Caucasian male, who died from head trauma. The patient had a history of asthma. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0155] LUNGFET03 Library was constructed using 1 microgram of polyA RNA isolated from lung tissue removed from a Caucasian female fetus, who died at 20 weeks of gestation from fetal demise. Serology was negative. Family history included seven days of erythromycin treatment for bronchitis in the mother during the first trimester. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte). COLNFET02 (colon), LIVRFET02 (liver), and LUNGFET05 (lung) are other libraries from the same donor.

[0156] LUNGNOT01 Library was constructed at Stratagene, using RNA isolated from the lung tissue of a 72-year-old male. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector. Following Lambda UniZAP packaging, 2×10e6 primary clones were then amplified to stabilize the library for long-term storage. Amplification may significantly skew sequence abundances. The same Stratagene library (STR937210) was used for LUNGNOM01, obtained from the WashU-Merck EST Project.

[0157] LUNGNOT02 Library was constructed using RNA isolated from the lung tissue of a 47-year-old Caucasian male, who died of a subarachnoid hemorrhage. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0158] LUNGNOT04 Library was constructed using 1.6 micrograms of polyA RNA isolated from the lung tissue of a 2-year-old Hispanic male, who died from cerebral anoxia. Past medical history and serologies were negative. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0159] LUNGNOT09 Library was constructed using 1 microgram of polyA RNA isolated from the lung tissue of a 23-week-old Caucasian male fetus. The pregnancy was terminated following a diagnosis by ultrasound of infantile polycystic kidney disease. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0160] LUNGNOT10 Library was constructed using 1 microgram of polyA RNA isolated from the lung tissue of a Caucasian male fetus, who died at 23 weeks of gestation from premature birth. Serology was negative. Family history included diabetes in the mother. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0161] LUNGNOT12 Library was constructed using 0.464 micrograms of polyA RNA isolated from nontumorous lung tissue removed from a 78-year-old Caucasian male during a segmental lung resection and regional lymph node resection. Surgery followed a diagnosis of a malignant neoplasm of the right upper lobe. Pathology indicated fibrosis pleura was puckered, but not invaded. Pathology for the associated tumor tissue indicated an invasive pulmonarygrade 3 adenocarcinoma, forming a peripheral mass with associated fibrosis. The patient presented with premature ventricular beats. Patient history included cerebrovascular disease, arteriosclerotic coronary artery disease, thrombophlebitis, chronic obstructive pulmonary disease, asthma, and tobacco use. Previous surgeries included a cholecystectomy, radical prostatectomy, and regional lymph node excision for malignant prostate neoplasm. Patient medications included Cipro I.V. (ciprofloxacin) for a systemic infection; Atenolol (tenormin) for arrhythmia; Darvocet-N (propoxyphene napsylate) for pain; Naprosyn (naproxen), an anti-inflammatory and analgesic; and multivitamins. Family history included intracranial hematoma with deep coma following injury in the mother, and cerebrovascular disease, arteriosclerotic coronary artery disease, and Type I diabetes in a sibling. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0162] LUNGNOT15 Library was constructed using 1 microgram of polyA RNA isolated from nontumorous lung tissue removed from the left lower lobe of a 69-year-old Caucasian male during a segmental lung resection. Pathology for the associated tumor tissue indicated residual grade 3 invasive squamous cell carcinoma. The upper lobe also contained residual grade 3 invasive squamous cell carcinoma. Surgical margins and lymph nodes were negative for tumor. Patient history included acute myocardial infarction, prostatic hyperplasia, benign hypertension, malignant skin neoplasm, and tobacco use. Previous surgeries included a multivessel coronary artery bypass. Patient medications included Hytrin (terazosin) for benign prostate hyperplasia; Norvasc (amiodipine besylate) for angina; Atenolol (tenormin) for arrhythmia; KCL (potassium chloride); Lasix (furosemide), a diuretic; and blood-pressure medicine. Family history included cerebrovascular disease and Type I diabetes in the mother, and acute myocardial infarction and arteriosclerotic coronary disease in the father. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0163] SINTBST01 Library was constructed using 1 microgram of polyA RNA isolated from the ileum tissue of an 18-year-old Caucasian female with irritable bowel syndrome (IBS). The ileum tissue, along with the cecum and appendix, were removed during bowel anastomosis. Pathology indicated Crohn's disease of t he ileum, involving 15 cm of the small bowel. The cecum and appendix were unremarkable, and the margins were uninvolved. The patient presented with abdominal pain and regional enteritis. Patient history included osteoporosis of the vertebra and abnormal blood chemistry. Patient medications included Prilosec (omeprazole), Pentasa (mesalamine), amoxicillin, and multivitamins. Family history included cerebrovascular disease in the mother and a grandparent, and atherosclerotic coronary artery disease in a grandparent. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0164] LUNGNOT18 Library was constructed using 1 microgram of polyA RNA isolated from nontumorous lung tissue removed from the left upper lobe of a 66-year-old Caucasian female during a segmental lung resection and regional lymph node biopsy. Pathology for the associated tumor tissue indicated a grade 2 adenocarcinoma with bronchoalveolar features and prominent inflammation, forming a well-circumscribed nodular mass. The tumor did not involve the pleura. Surgical margins and lymph nodes were negative for tumor. Patient history included cerebrovascular disease, atherosclerotic coronary artery disease, pulmonary insufficiency, and a normal delivery. Previous surgeries included an endarterectomy. Patient medications included Trental, Zocor, and aspirin. Family history included a myocardial infarction in the mother and father, and atherosclerotic coronary artery disease in a sibling. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0165] LUNGNOT20 Library was constructed using 1 microgram of polyA RNA isolated from lung tissue removed from the right upper lobe a 61-year-old Caucasian male during a segmental lung resection. Pathology indicated panacinal emphysema with blebs in the right anterior upper lobe and apex, as well as emphysema in the right posterior upper lobe. Panacinal emphysema was also identified in the left upper lobe lingula with one 3 mm calcified granuloma. Patient history included benign hypertension, angina pectoris, gastric ulcer, and tobacco and alcohol use. Patient medications included Azmacort, Atrovent, Ventolin, nitroglycerine, and tetracycline HCl. Family history included a subdural hemorrhage in the father; cancer of an unidentified site in the mother; benign hypertension, atherosclerotic coronary artery disease, pneumonia, and an unspecified muscle disorder in a sibling; and benign hypertension, atherosclerotic coronary artery disease, and cancer of an unidentified site in a grandparent. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0166] MMLR1DT01 Library was constructed using 2 micrograms of polyA RNA isolated from adherent mononuclear cells, which came from a pool of male and female donors. The cells were cultured for 24 hours following Ficoll Hypaque centrifugation. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0167] MMLR3DT01 Library was constructed using 2 micrograms of polyA RNA isolated from adherent mononuclear cells, which came from a pool of male and female donors. The cells were cultured for 72 hours following Ficoll Hypaque centrifugation. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0168] MPHGLPT02 Library was constructed using 1 microgram of polyA RNA isolated from adherent mononuclear cells, which came from a pool of male and female donors. The cells were isolated using Ficoll Hypaque centrifugation, and the predominantly macrophage-containing population was stimulated with LPS at 1 μg/ml for 2 hours before isolation of total RNA and polyA selection. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0169] MPHGNOT02 Library was constructed using total RNA isolated from plastic adherent mononuclear cells obtained from the peripheral blood of a healthy 24-year-old Caucasian male. After a 2-hour culture in medium containing normal human serum, the cells were harvested and lysed in a buffer containing GuSCN, then spun through CsCl to obtain RNA for library construction. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector. Because this library was made from total RNA, it has an unusually high proportion of unique singleton sequences, which may not all come from polyA RNA species.

[0170] MPHGNOT03 Library was constructed using 4 micrograms of polyA RNA isolated from plastic adherent (2 hour culture) mononuclear cells isolated from buffy coat units obtained from unrelated male and female donors. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0171] NEUTFMT01 Library was constructed using total RNA isolated from peripheral blood granulocytes collected by density gradient centrifugation through Ficoll-Hypaque. The cells were isolated from buffy coat units obtained from unrelated male and female donors. Cells were cultured in 10 nM fMLP for 30 minutes, lysed in GuSCN, and spun through CsCl to obtain RNA for library construction. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector. Because this library was made from total RNA, it has an unusually high proportion of unique singleton sequences, which may not all come from polyA RNA species.

[0172] NEUTGMT01 Library was constructed using 1 microgram of polyA RNA isolated from peripheral blood granulocytes collected by density gradient centrifugation through Ficoll-Hypaque. The cells were isolated from buffy coat units obtained from 20 unrelated male and female donors. Cells were cultured in 10 nM GM-CSF for 1 hour before washing and harvesting for total RNA preparation. cDNA synthesis was initiated using a NotI-oligo(dT)primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0173] NEUTGMT01 Library was constructed using 1 microgram of polyA RNA isolated from peripheral blood granulocytes collected by density gradient centrifugation through Ficoll-Hypaque. The cells were isolated from buffy coat units obtained from 20 unrelated male and female donors. Cells were cultured in 10 nM GM-CSF for 1 hour before washing and harvesting for total RNA preparation. cDNA synthesis was initiated using a NotI-oligo(dT)primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0174] NEUTLPT01 Library was constructed using 64 micrograms of total RNA isolated from peripheral blood granulocytes collected by density gradient centrifugation through Ficoll-Hypaque. The cells were isolated from buffy coat units obtained from unrelated male and female donors. Cells were cultured in 100 ng/ml E. coliLPS for 30 minutes, lysed in GuSCN, and spun through CsCl to obtain RNA for library construction. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector. Because this library was made from total RNA, it has an unusually high proportion of unique singleton sequences, which may not all come from polyA RNA species.

[0175] PANCDIT01 Library was constructed using polyA RNA isolated from pancreas tissue removed from a 15-year-old Caucasian male, who died from a self-inflicted gunshot wound. Patient history included Type I diabetes. Previous surgeries included an appendectomy. Patient medications included insulin. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0176] PANCNOT01 Library was constructed using RNA isolated from the pancreatic tissue of a 29-year-old Caucasian male, who died from head trauma. Serologies were positive for cytomegalovirus (CMV) but otherwise negative. Patient history included alcohol, marijuana, and tobacco use. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0177] PANCNOT04 Library was constructed using 2.5 micrograms of polyA RNA isolated from the pancreatic tissue of a 5-year-old Caucasian male, who died in a motor vehicle accident. Serology was positive for cytomegalovirus (CMV), but otherwise serologies and medical history were negative. Patient medications included Dopamine, Dobutamine, Ancef (cefazolin), DDAVP, Mannitol, and Pepcid (famotidine). cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0178] PANCNOT04 Library was constructed using 2.5 micrograms of polyA RNA isolated from the pancreatic tissue of a 5-year-old Caucasian male, who died in a motor vehicle accident. Serology was positive for cytomegalovirus (CMV), but otherwise serologies and medical history were negative. Patient medications included Dopamine, Dobutamine, Ancef (cefazolin), DDAVP, Mannitol, and Pepcid (famotidine). cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0179] PANCNOT05 Library was constructed using 1.6 micrograms of polyA RNA isolated from the pancreatic tissue of a 2-year-old Hispanic male, who died from cerebral anoxia. Past medical history and serologies were negative. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0180] SINTNOT13 Library was constructed using 1 microgram of polyA RNA isolated from ileum tissue removed from a 25-year-old Asian female during a partial colectomy and temporary ileostomy. Pathology indicated moderately active chronic ulcerative colitis, involving colonic mucosa from the distal margin to the ascending colon. The cecum, appendix, and attached ileal segment were uninvolved. Rectal mucosa also showed moderately active chronic ulcerative colitis. The patient presented with diarrhea, blood in the stool, and abdominal pain. Patient history included papilloma virus, deficiency anemia, migraines, backache, depressive disorder, and tobacco use. Patient medications included Rowasa (mesalamine rectal suppositories) for colitis, Nordette(levonorgestrel and ethinyl estradiol), Asacol (mesalamine), and digestive herbs. Family history included hyperlipidemia and depressive disorder in the mother; and a malignant cervical neoplasm, viral hepatitis A, depressive disorder, and alcohol and drug use in the sibling(s). cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0181] SPLNFET01 Library was constructed at Stratagene, using RNA isolated from a pool of fetal spleen tissue. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector. Following Lambda UniZAP packaging, 2×10e6 primary clones were then amplified to stabilize the library for long-term storage. Amplification may significantly skew sequence abundances. The same Stratagene library (STR937205) was used for SPLNFEM01, obtained from the WashU-Merck EST Project.

[0182] SPLNFET02 Library was constructed using 1 microgram of polyA RNA isolated from spleen tissue removed from a Caucasian male fetus, who died at 23 weeks of gestation from premature birth. Serology was negative. Family history included diabetes in the mother. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0183] SPLNNOT02 Library was constructed using RNA isolated from the spleen tissue of a 29-year-old Caucasian male, who died from head trauma. Serologies were positive for cytomegalovirus (CMV) but otherwise negative. Patient history included alcohol, marijuana, and tobacco use. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0184] SPLNNOT04 Library was constructed using 1 microgram of polyA RNA isolated from the spleen tissue of a 2-year-old Hispanic male, who died from cerebral anoxia. Past medical history and serologies were negative. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0185] SYNOOAT01 Library was constructed using 1 microgram of polyA RNA isolated from the knee synovial membrane tissue of an 82-year-old female with osteoarthritis. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0186] SYNORAB01 Library was constructed using RNA isolated from the synovial membrane tissue of a 68-year-old Caucasian female with rheumatoid arthritis. Patient medications included enteric coated ASA, fluoride 20, fiorinal, iron gluconate, Gold 1 tab, and multivitamins. cDNA synthesis was initiated using a combination of oligo(dT) and random priming. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0187] SYNORAT01 Library was constructed using RNA isolated from synovial membrane tissue removed from the elbow of a 51-year-old Asian female with rheumatoid arthritis. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0188] SYNORAT03 Library was constructed using 1 microgram of polyA RNA isolated from the wrist synovial membrane tissue of a 56-year-old female with rheumatoid arthritis. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0189] SYNORAT04 Library was constructed using 1 microgram of polyA RNA isolated from the wrist synovial membrane tissue of a 62-year-old female with rheumatoid arthritis. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0190] SYNORAT05 Library was constructed using 1 microgram of polyA RNA isolated from the knee synovial tissue of a 62-year-old female with rheumatoid arthritis. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0191] TBLYNOT01 Library was constructed at Stratagene (STR937214), using RNA isolated from a hybrid of T-B lymphoblasts from a leukemic cell line. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector. Following Lambda UniZAP packaging, 2×10e6 primary clones were then amplified to stabilize the library for long-term storage. Amplification may significantly skew sequence abundances.

[0192] THP1AZT01 Library was constructed using 1 microgram of polyA RNA isolated from THP-1 promonocyte cells treated for three days with 0.8 micromolar 5-aza-2′-deoxycytidine. THP-1 (ATCC TIB 202) is a human promonocyte line derived from peripheral blood of a 1-year-old Caucasian male with acute monocytic leukemia (ref: Int. J. Cancer(1980) 26:171). cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0193] THP1AZT01 Library was constructed using 1 microgram of polyA RNA isolated from THP-1 promonocyte cells treated for three days with 0.8 micromolar 5-aza-2′-deoxycytidine. THP-1 (ATCC TIB 202) is a human promonocyte line derived from peripheral blood of a 1-year-old Caucasian male with acute monocytic leukemia (ref: Int. J. Cancer(1980) 26:171). cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0194] THP1NOB0l Library was constructed using RNA isolated from cultured, unstimulated THP-1 cells. THP-1 (ATCC TIB 202) is a human promonocyte line derived from the peripheral blood of a 1-year-old Caucasian male with acute monocytic leukemia (ref: Int. J. Cancer(1980) 26:171). RNA was isolated from 2×108 cells using GuSCN lysis, followed by DNAse treatment. cDNA synthesis was initiated using a combination of oligo(dT) and random priming. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0195] THP1PLB01 Library was constructed using RNA isolated from THP-1 cells cultured for 48 hours with 100 ng/ml phorbol ester (PMA), followed by a 4-hour culture in media containing 1 ug/ml LPS. THP-1 (ATCC TIB 202) is a human promonocyte line derived from the peripheral blood of a 1-year-old male with acute monocytic leukemia (ref: Int. J. Cancer(1980) 26:171). cDNA synthesis was initiated using a combination of oligo(dT) and random priming. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0196] THP1PLB02 Library was constructed by reamplification of THP1PLB01, which was made using RNA isolated from THP-1 cells cultured for 48 hours with 100 ng/ml phorbol ester (PMA), followed by a 4-hour culture in media containing 1 ug/ml LPS. THP-1 (ATCC TIB 202) is a human promonocyte line derived from the peripheral blood of a 1-year-old male with acute monocytic leukemia (ref: Int. J. Cancer(1980) 26:171). cDNA synthesis was initiated using a combination of oligo(dT) and random priming. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the-XhoI and EcoRI sites of the Lambda UniZAP vector. Following Lambda UniZAP packaging, 1×10e6 primary clones were then amplified to stabilize the library for long-term storage. Amplification may significantly skew sequence abundances.

[0197] THP1NOT01 Library was constructed using 1 microgram of polyA RNA isolated from untreated THP-1 cells. THP-1 (ATCC TIB 202) is a human promonocyte line derived from the peripheral blood of a 1-year-old Caucasian male with acute monocytic leukemia (ref: Int. J. Cancer(1980) 26:171).cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0198] THP1NOT03 Library was constructed using 1 microgram of polyA RNA isolated from untreated THP-1 cells. THP-1 (ATCC TIB 202) is a human promonocyte line derived from the peripheral blood of a 1-year-old Caucasian male with acute monocytic leukemia (ref: Int. J. Cancer(1980) 26:171). cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).

[0199] THP1PEB01 Library was constructed using RNA isolated from THP-1 cells cultured for 48 hours with 100 ng/ml phorbol ester (PMA). Following culture in PMA, cells were lysed in GuSCN. THP-1 (ATCC TIB 202) is a human promonocyte line derived from the peripheral blood of a 1-year-old Caucasian male with acute monocytic leukemia (ref: Int. J. Cancer(1980) 26:171). cDNA synthesis was initiated using a combination of oligo(dT) and random priming. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0200] THYMNOT02 Library was constructed using polyA RNA isolated from thymus tissue removed from a 3-year-old Caucasian male, who died from drowning. Serologies were negative. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0201] TLYMNOR01 Library was constructed using RNA isolated from non-adherent peripheral blood mononuclear cells obtained from a 24-year-old Caucasian male. (This is the same RNA source used for TLYMNOT01.) The cells were purified on Ficoll Hypaque, then harvested, lysed in GuSCN, and spun through CsCl to obtain RNA for library construction. cDNA synthesis was initiated using a random primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0202] TLYMNOT01 Library was constructed using RNA isolated from non-adherent peripheral blood mononuclear cells obtained from a 24-year-old Caucasian male. The cells were purified on Ficoll Hypaque, then harvested, lysed in GuSCN, and spun through CsCl to obtain RNA for library construction. PolyA RNA was isolated using oligo(dT) cellulose. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector. TLYMNOR01 is a random-primed library from the same RNA source.

[0203] TMLR2DT01 Library was constructed using RNA isolated from non-adherent peripheral blood mononuclear cells. The blood was obtained from unrelated male and female donors. Cells from each donor were purified on Ficoll Hypaque, then co-cultured for 24 hours in medium containing normal human serum at a cell density of 2×106 cells/ml. cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0204] TMLR3DT01 Library was constructed using RNA isolated from non-adherent and adherent peripheral blood mononuclear cells collected from two unrelated Caucasian male donors (25 and 29 years old). Cells from each donor were purified on Ficoll Hypaque, then co-cultured for 96 hours in medium containing normal human serum at a cell density of 2×106 cells/ml. The non-adherent cells were collected, then the adherent cells were collected by scraping with a rubber policeman, and the populations were pooled. The pooled cells were washed once in PBS, lysed in a buffer containing GuSCN, and spun through CsCl to obtain RNA for library construction. PolyA RNA was isolated using a Qiagen Oligotex kit. cDNA synthesis was initiated using an Xhol-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector.

[0205] TONSNOT01 Library was constructed using 1.2 micrograms of polyA RNA isolated from the tonsil tissue of a 6-year-old Caucasian male with lymphoid hyperplasia of the tonsils. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to SalI adaptors, digested with NotI, size-selected, and cloned into the NotI and SalI sites of the pSPORT1 vector.

[0206] U937NOT01 Library was constructed at Stratagene (STR937207), using RNA isolated from the U937 monocyte-like cell line. This line (ATCCCRL1593) was established by C. Sundstrom and K. Nilsson in 1974 from malignant cells obtained from the pleural effusion of a 37-year-old Caucasian male with diffuse histiocytic lymphoma (ref: Int. J. Cancer(1976) 17: 565-577). cDNA synthesis was initiated using an XhoI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with XhoI, size-selected, and cloned into the XhoI and EcoRI sites of the Lambda UniZAP vector. Following Lambda UniZAP packaging, 2×10e6 primary clones were then amplified to stabilize the library for long-term storage. Amplification may significantly skew sequence abundances.

[0207] UCMCL5T01 Library was constructed using 1 microgram of polyA RNA isolated from mononuclear cells obtained from the umbilical cord blood of 12 individuals. The.cells were cultured for 12 days with IL-5 before RNA was obtained from the pooled lysates. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pBluescript vector.

[0208] UCMCNOT02 Library was constructed using 1 microgram of polyA RNA isolated from mononuclear cells obtained from the umbilical cord blood of nine individuals. cDNA synthesis was initiated using a NotI-oligo(dT) primer. Double-stranded cDNA was blunted, ligated to EcoRI adaptors, digested with NotI, size-selected, and cloned into the NotI and EcoRI sites of the pINCY vector (Incyte).