Abstract:
The present invention includes methods of normalizing quantitative and non-quantitative nucleic acid detection assays by identifying genes whose expression level is invariant among cell or tissue types. The methods of the invention can be used in the diagnosis of disease, in quality control in evaluating external data or databases, and in normalization of external data for comparative purposes. The genes of the invention can be used to produce microarrays that generate data with improved reliability.

Description:
RELATED APPLICATIONS  
       [0001]    This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60/399,158, filed Jul. 30, 2002, which is herein incorporated by reference in its entirety. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The invention relates generally to control genes that maybe utilized for normalizing hybridization and/or amplification reactions, as well as methods of identifying these genes that may be used in toxicology studies and in analyzing gene expression data sets for quality and compatibility with other data sets.  
         BACKGROUND OF THE INVENTION  
         [0003]    Nucleic acid hybridization and other quantitative nucleic acid detection assays are routinely used in medical and biotechnological research and development, diagnostic testing, drug development and forensics. Such technologies have been used to identify genes which are up- or down-regulated in various disease or physiological states, to analyze the roles of the members of cellular signaling cascades and to identify drugable targets for various disease and pathology states.  
           [0004]    Examples of technologies commonly used for the detection and/or quantification of nucleic acids include Northern blotting (Krumlauf (1994),  Mol Biotechnol  2: 227-242), in situ hybridization (Parker &amp; Barnes (1999),  Methods Mol Biol  106: 247-283), RNAse protection assays (Hod (1992),  Biotechniques  13: 852-854; Saccomanno et al. (1992),  Biotechniques  13: 846-850), microarrays, and reverse transcription polymerase chain reaction (RT-PCR) (see Bustin (2000),  J Mol Endocrin  25: 169-193).  
           [0005]    The reliability of these nucleic acid detection methods depend on the availability of accurate means for accounting for variations between analyses. For example, variations in hybridization conditions, label intensity, reading and detector efficiency, sample concentration and quality, background effects, and image processing effects each contribute to signal heterogeneity (Hegde et al. (2000),  Biotechniques  29: 548-562; Berger et al. (2000), WO 00/04188). Normalization procedures used to overcome these variations often rely on control hybridizations to housekeeping genes such as P-actin, glyceraldehyde-3-phosphate dehydrogenase (GADPH), and the transferrin receptor gene (Eickhoff et al. (1999),  Nucl Acids Res  27:e33; Spiess et al. (1999),  Biotechniques  26: 46-50. These methods, however, generally do not provide the signal linearity sufficient to detect small but significant changes in transcription or gene expression (Spiess et al.(1999),  Biotechniques  26:46-50). In addition, the steady state levels of many housekeeping genes are susceptible to alterations in expression levels that are dependent on cell differentiation, nutritional state, specific experimental and stimulation protocols (Eickhoff et al. (1999),  Nucl Acids Res  27:e33; Spiess et al. (1999),  Biotechniques  26:46-50; Hegde et al. (2000),  Biotechniques  29:548-562; and Berger et al. (2000), WO 00/04188). Consequently, there exists a need for the identification and use of additional genes that may serve as effective controls in nucleic acid detection assays.  
         SUMMARY OF THE INVENTION  
         [0006]    The present invention includes methods of identifying at least one gene that is consistently expressed across different cell or tissue types in an organism, comprising: preparing gene expression profiles for different cell or tissue types from the organism; calculating a coefficient of variation for at least one gene in each of the profiles across the different cell or tissue types; and selecting any gene whose coefficient of variation indicates that the gene is consistently expressed across the different cell or tissue types. The coefficient of variation may be less than about 40% and the methods may comprise creating gene expression profiles for about 10, 25, 50, 100 or more different cell or tissue types. The gene expression profiles may be prepared be querying a gene expression database.  
           [0007]    The invention also includes a set of probes comprising at least two probes that specifically hybridize to a control gene identified by the methods of the invention. Such sets of probes may comprise probes that specifically hybridize to at least about 10, 25, 50 or 100 control genes. In some formats, the sets of probes are attached to a solid substrate such as a microarray or chip.  
           [0008]    The invention also includes methods of normalizing the data from a nucleic acid detection assay comprising: detecting the expression level for at least one gene in a nucleic acid sample; and normalizing the expression of said at least one gene with the detected expression of at least one control gene identified by the method of the invention. The number of control genes used to normalize gene expression data may comprise about 10, 25, 50, 100 or more of the control genes herein identified.  
           [0009]    In another embodiment, the invention includes a set of probes comprising at least two probes that specifically hybridize to a gene of Table 1. The set may comprise at least about 10, 25, 50, 100 or more the control genes of Table 1. The sets of probes may or may not be attached to a solid substrate such as a chip.  
           [0010]    The invention, in another embodiment, includes methods of normalizing the data from a nucleic acid detection assay comprising: detecting the expression level for at least one gene in a nucleic acid sample; and normalizing the expression of said at least one gene with the detected expression of at least one control gene of Table 1. The number of control genes used to normalize gene expression data may comprise about 10, 25, 50, 100, 500 or more of the control genes herein identified.  
         DETAILED DESCRIPTION  
         [0011]    The present Inventors have identified rat control genes that may be monitored in nucleic acid detection assays and whose expression levels may be used to normalize gene expression data or evaluate the suitability of test data to compare to or to include in a database of like data. Normalization of gene expression data from a cell or tissue sample with the expression level(s) of the identified control genes allows the accurate assessment of the expression level(s) for genes that are differentially regulated between samples, tissues, treatment conditions, et. These control genes may be used across a broad spectrum of assay formats, but are particularly useful in microarray or hybridization based assay formats.  
           [0012]    A. Nucleic Acid Detection Assay Controls  
           [0013]    1. Selection of Control Genes  
           [0014]    As used herein, the genes selected by the disclosed methods as well as the rat genes and nucleic acids of Table 1 are referred to as “invariant” or “control genes.” Control genes of the invention may be produced by a method comprising preparing gene expression profiles (a representation of the expression level for at least one gene, preferably 10, 25, 50, 100, 500 or more, or, most preferably, nearly all or all expressed genes in a sample) from at least two (or a variety) of cell or tissue types, or from a set of samples of at least one cell or tissue type in which the set contains normal samples (from healthy animals), disease state samples, toxin-exposed samples, etc., measuring the level of expression for at least one gene in each of the gene expression profiles to produce gene expression data, calculating a coefficient of variation in the expression level from the gene expression data for each gene (% CV) and selecting genes whose coefficient of variation indicates that the gene is consistently expressed at about the same level in the different cell or tissue types. In one embodiment, such genes that are expressed at about the same level, or are invariantly expressed, are those genes that have a coefficient of variation (expressed as a percentage) of less than or equal to about 40%.  
           [0015]    In the methods of the invention, gene expression profiles maybe produced by any means of quantifying gene expression for at least one gene in the tissue or cell sample. In preferred methods, gene expression is quantified by a method selected from the group consisting of a hybridization assay or an amplification assay. Hybridization assays may be based on any assay format that relies on the hybridization of a probe or primer to a nucleic acid molecule in the sample. Such formats include, but are not limited to, differential display formats and microarray hybridization, including microarrays produced in chip format. Amplification assays include, but are not limited to, quantitative PCR, semiquantitative PCR and assays that rely on amplification of nucleic acids subsequent to the hybridization of the nucleic acid to a probe or primer. Such assays include the amplification of nucleic acid molecules from a sample that are bound to a microarray or chip.  
           [0016]    In other circumstances, gene expression profiles may be produced by querying a gene expression database comprising expression results for genes from various cell or tissue samples. The gene expression results in the database may be produced by any available method, such as differential display methods and micro array-based hybridization methods. The gene expression profile is typically produced by the step of querying the database with the identity of a specific cell or tissue type for the genes that are expressed in the cell or tissue type and/or the genes that are differentially regulated compared to a control cell or tissue sample. Available databases include, but are not limited to, the Gene Logic Gene Express™ database, the Gene Expression Omnibus gene expression and hybridization array repository available through NCBI (www.ncbi.nlm.nih.gov/entrez) and the SAGE™ gene expression database.  
           [0017]    In preferred embodiments, the statistical measure referred to herein as the coefficient of variation (% CV) is calculated on a gene by gene basis across a number of samples or across a reference database to find the least variant genes with respect to a number of cell or tissue types or sample treatments.  
           [0018]    Further, the statistical methods of the invention are particularly useful for determining the compatibility of a test sample to an entire set of samples, or an existing database derived from those samples. For instance, a % CV value for genes that have been shown to be the most resistant to variability is calculated for all samples within a test group or test database. These % CV values are then compared to those from a standard reference database. Accordingly, a closeness distribution of all individual samples in the test database to the reference database as a whole can be generated to evaluate the compatibility of new samples. The genes identified in Table I show invariant patterns of expression and can be used to assess compatibility and reliability of gene expression experiments and predictive modeling experiments. These genes show low variability both in control groups from many different experiments and in studies of disruptions of gene expression, such as those occurring in disease states. As a result, these genes can be used as an internal standard for comparing gene expression data. Measurements of expression levels of these genes are used to determine the extent of compatibility of data from different sources and the need, or lack thereof, for normalization or further quality control and adjustments. These measurements also provide an internal standard that supplies a reference point for highly disrupted patterns of gene expression. These genes are also of critical importance for determining relative expression if small numbers of markers are used in custom microarrays.  
           [0019]    The cell or tissue sample that reduced to prepare gene expression profiles may include any cell or tissue sample available. Such samples include, but are not limited to, tissues removed as surgical samples, diseased or normal tissues, in vitro or in vivo grown cells, and cell cultures and cells or tissues from animals exposed to an agent such as a toxin. The number of samples that may be used to calculate a coefficient of variation is variable, but may include about 3, 10, 25, 50, 100, 200, 500 or more cell or tissue samples. The cell or tissue samples may be derived from an animal or plant, preferably a mammal, most preferably a rat. In some instances, the cell or tissue samples may be human, canine (dog), mouse or rat in origin.  
           [0020]    In some embodiments of the invention, the coefficient of variation maybe calculated from raw expression data or from data that has been normalized to control for the mechanics of hybridization, such as data normalized or controlled for background noise due to non-specific hybridization. Such data typically includes, but is not limited to, fluorescence readings from microarray based hybridizations, densitometry readings produced from assays that rely on radiological labels to detect and quantify gene expression and data produced from quantitative or semi-quantitative amplification assays.  
           [0021]    The coefficient of variation (CV) is typically calculated by calculating a mean value for the expression level of a given gene across a number of samples and calculating the standard deviation (SD) from that mean. The CV may be calculated by the following equation: CV=SD/Mean and may or may not be presented as a percentile value. Genes with a CV of less than about 40% may be selected as control genes or are considered as genes that are consistently expressed across the different cell or tissue types tested.  
           [0022]    As used herein, “background” refers to signals associated with non-specific binding (cross-hybridization). In addition to cross-hybridization, background may also be produced by intrinsic fluorescence of the hybridization format components themselves.  
           [0023]    “Bind(s) substantially” refers to complementary hybridization between an oligonucleotide probe and a nucleic acid sample and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the nucleic acid sample.  
           [0024]    The phrase “hybridizing specifically to” refers to the binding, duplexing or hybridizing of a molecule substantially to or only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.  
           [0025]    2. Preparation of Controls Genes, Probes and Primers  
           [0026]    The control genes listed in Table I may be obtained from a variety of natural sources such as organisms, organs, tissues and cells. The sequences of known genes are in the public databases. The GenBank Accession Number corresponding to the Normalization Control Genes can be found in Table 1. The sequences of the genes in GenBank (http://www.ncbi.nlm.nih.gov/) are herein incorporated by reference in their entirety as of the priority date of this application.  
           [0027]    Probes or primers for the nucleic acid detection assays described herein that specifically hybridize to a control gene may be produced by any available means. For instance, probe sequences may be prepared by cleaving DNA molecules produced by standard procedures with commercially available restriction endonucleases or other cleaving agents. Following isolation and purification, these resultant normalization control gene fragments can be used directly, amplified by PCR methods or amplified by replication or expression from a vector.  
           [0028]    Control genes and control gene probes or primers (i.e., synthetic oligonucleotides and polynucleotides) are most easily synthesized by chemical techniques, for example, the phosphoramidite method of Matteucci et al. ((1981)  J Am Chem Soc  103:3185-3191) or using automated synthesis methods using the GenBank sequences disclosed in Table 1. Probes for attachment to microarrays or for use as primers in amplification assays may be produced from the sequences of the genes identified herein using any available software, including, for instance, software available from Molecular Biology Insights, Olympus Optical Co. and Premier Biosoft International.  
           [0029]    In addition, larger nucleic acids can readily be prepared by well known methods, such as synthesis of a group of oligonucleotides that define various modular segments of the normalization control genes and normalization control gene segments, followed by ligation of oligonucleotides to build the complete nucleic acid molecule.  
           [0030]    B. Normalization Methods  
           [0031]    Gene expression data produced from the control genes in a given sample or samples may be used to normalize the gene expression data from other genes using any available arithmatic or calculative means. In particular, gene expression data from the control genes in Table 1 are useful to normalize gene expression data for toxicology testing or modeling in an animal model, preferably in a rat. Such methods include, but are not limited, methods of data analysis described by Hegde et al. (2000),  Biotechniques  29:548-562; Winzeller et al. (1999),  Meth Enzymol  306:3-18; Tkatchenko et al. (2000),  Biochimica et Biophysica Acta  1500:17-30; Berger et al. (2000), WO 00/04188; Schuchhardt et al. (2000),  Nucl Acids Res  28:e47; Eickhoffet al. (1999),  Nucl Acids Res  27:e33. Micro-array data analysis and image processing software packages and protocols, including normalization methods, are also available from BioDiscovery (http://www.biodiscovery.com), Silicon Graphics (http://www.sigenetics.com), Spotfire (http://www.spotfire.com), Stanford University (http://rana.Stanford.EDU/software), National Human Genome Research Institute (http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/img_analysis.html), TIGR (http://www.tigr.org/softlab), and Affymetrix (affy and maffy packages), among others.  
           [0032]    C. Assay or Hybridization Formats  
           [0033]    The control genes of the present invention may be used in any nucleic acid detection assay format, including solution-based and solid support-based assay formats. As used herein, “hybridization assay format(s)” refer to the organization of the oligonucleotide probes relative to the nucleic acid sample. The hybridization assay formats that may be used with the control genes and methods of the present invention include assays where the nucleic acid sample is labeled with one or more detectable labels, assays where the probes are labeled with one or more detectable labels, and assays where the sample or the probes are immobilized. Hybridization assay formats include but are not limited to: Northern blots, Southern blots, dot blots, solution-based assays, branched DNA assays, PCR, RT-PCR, quantitative or semi-quantitative RT-PCR, microarrays and biochips.  
           [0034]    As used herein, “nucleic acid hybridization” simply involves contacting a probe and nucleic acid sample under conditions where the probe and its complementary target can form stable hybrid duplexes through complementary base pairing (see Lockhart et al., (1999) WO 99/32660). The nucleic acids that do not form hybrid duplexes are then washed away leaving the hybridized nucleic acids to be detected, typically through detection of an attached detectable label.  
           [0035]    It is generally recognized that nucleic acids are denatured by increasing the temperature or decreasing the salt concentration of the buffer containing the nucleic acids. Under low stringency conditions (e.g., low temperature and/or high salt) hybrid duplexes (e.g., DNA-DNA, RNA-RNA or RNA-DNA) will form even where the annealed sequences are not perfectly complementary. Thus, specificity of hybridization is reduced at lower stringency. Conversely, at higher stringency (e.g., higher temperature or lower salt) successful hybridization requires fewer mismatches. One of skill in the art will appreciate that hybridization conditions may be selected to provide any degree of stringency. In a preferred embodiment, hybridization is performed at low stringency, in this case in 6×SSPE-T at 37° C. (0.005% Triton X-100) to ensure hybridization, and then subsequent washes are performed at higher stringency (e.g., 1×SSPE-T at 37° C.) to eliminate mismatched hybrid duplexes. Successive washes may be performed at increasingly higher stringency (e.g., down to as low as 0.25×SSPE-T at 37° C. to 50° C. until a desired level of hybridization specificity is obtained. Stringency can also be increased by addition of agents such as formamide. Hybridization specificity may be evaluated by comparison of hybridization to the test probes with hybridization to the various controls that can be present (e.g., expression level control, normalization control, mismatch controls, etc.).  
           [0036]    As used herein, the term “stringent conditions” refers to conditions under which a probe will hybridize to a complementary control nucleic acid, but with only insubstantial hybridization to other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.  
           [0037]    Typically, stringent conditions will be those in which the salt concentration is at least about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.  
           [0038]    In general, there is a tradeoff between hybridization specificity (stringency) and signal intensity. Thus, in a preferred embodiment, the wash is performed at the highest stringency that produces consistent results and that provides a signal intensity greater than approximately 10% of the background intensity. Thus, in a preferred embodiment, the hybridized array may be washed at successively higher stringency solutions and read between each wash. Analysis of the data sets thus produced will reveal a wash stringency above that the hybridization pattern is not appreciably altered and which provides adequate signal for the particular oligonucleotide probes of interest.  
           [0039]    The “percentage of sequence identity” or “sequence identity” is determined by comparing two optimally aligned sequences or subsequences over a comparison window or span, wherein the portion of the polynucleotide sequence in the comparison window may optionally comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical residue (e.g., nucleic acid base or amino acid residue) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Percentage sequence identity when calculated using the programs GAP or BESTFIT (see below) is calculated using default gap weights. Sequences corresponding to the control genes of the invention may comprise at least about 70% sequence identity to those sequences identified by GenBank Accession Nos. in Table 1, preferably about 75%, 80% or 85% sequence identity, or more preferably, about 90%, 95% or more sequence identity.  
           [0040]    Homology or identity is determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al. (1990),  Proc Natl Acad Sci USA  87:2264-2268 and Altschul (1993),  J Mol Evol  36:290-300, fully incorporated by reference) which are tailored for sequence similarity searching. The approach used by the BLAST program is first to consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al. (1994),  Nat Genet  6: 119-129, which is fully incorporated by reference. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al. (1992),  Proc Natl Acad Sci USA  89:10915-10919, fully incorporated by reference). Four blastn parameters were adjusted as follows Q=10 (gap creation penalty) R=10 (gap extension penalty); wink=1 (generates word hits at every wink th  position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings were Q=9; R=2; wink=1; and gapw=32. A Bestfit comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty) and the equivalent settings in protein comparisons are GAP=8 and LEN=2.  
           [0041]    As used herein, a “probe” or “oligonucleotide probe” is defined as a nucleic acid, capable of binding to a nucleic acid sample or complementary control gene nucleic acid through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, U, C or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in probes may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.  
           [0042]    Probe arrays may contain at least two or more oligonucleotides that are complementary to or hybridize to one or more of the control genes described herein. Such arrays may also contain oligonucleotides that are complementary or hybridize to at least about 2, 3, 5, 7, 10, 50, 100 or more the genes described herein. Any solid surface to which oligonucleotides or nucleic acid sample can be bound, either directly or indirectly, either covalently or non-covalently, can be used. For example, solid supports for various hybridization assay formats can be filters, polyvinyl chloride dishes, silicon or glass based chips, etc. Glass-based solid supports, for example, are widely available, as well as associated hybridization protocols (see, e.g., Beattie, WO 95/11755).  
           [0043]    A preferred solid support is a high density array or DNA chip. This contains an oligonucleotide probe of a particular nucleotide sequence at a particular location on the array. Each particular location may contain more than one molecule of the probe, but each molecule within the particular location has an identical sequence. Such particular locations are termed features. There may be, for example, 2, 10, 100, 1000, 10,000, 100,000, 400,000, 1,000,000 or more such features on a single solid support. The solid support, or more specifically, the area wherein the probes are attached, may be on the order of a square centimeter.  
           [0044]    1. Dot Blots  
           [0045]    The control genes listed in Table I and methods of the present invention may be utilized in numerous hybridization formats such as dot blots, dipstick, branched DNA sandwich and ELISA assays. Dot blot hybridization assays provide a convenient and efficient method of rapidly analyzing nucleic acid samples in a sensitive manner. Dot blots are generally as sensitive as enzyme-linked immunoassays. Dot blot hybridization analyses are well known in the art and detailed methods of conducting and optimizing these assays are detailed in U.S. Pat. Nos. 6,130,042 and 6,129,828, and Tkatchenko et al. (2000),  Biochimica et Biophysica Acta  1500:17-30. Specifically, a labeled or unlabeled nucleic acid sample is denatured, bound to a membrane (i.e., nitrocellulose) and then contacted with unlabeled or labeled oligonucleotide probes. Buffer and temperature conditions can be adjusted to vary the degree of identity between the oligonucleotide probes and nucleic acid sample necessary for hybridization.  
           [0046]    Several modifications of the basic dot blot hybridization format have been devised. For example, reverse dot blot analyses employ the same strategy as the dot blot method, except that the oligonucleotide probes are bound to the membrane and the nucleic acid sample is applied and hybridized to the bound probes. Similarly, the dot blot hybridization format can be modified to include formats where either the nucleic acid sample or the oligonucleotide probe is applied to microtiter plates, microbeads or other solid substrates.  
           [0047]    2. Membrane-Based Formats  
           [0048]    Although each membrane-based format is essentially a variation of the dot blot hybridization format, several types of these formats are preferred. Specifically, the methods of the present invention may be used in Northern and Southern blot hybridization assays. Although the methods of the present invention are generally used in quantitative nucleic acid hybridization assays, these methods may be used in qualitative or semi-quantitative assays such as Southern blots, in order to facilitate comparison of blots. Southern blot hybridization, for example, involves cleavage of either genomic or cDNA with restriction endonucleases followed by separation of the resultant fragments on a polyacrylamide or agarose gel and transfer of the nucleic acid fragments to a membrane filter. Labeled oligonucleotide probes are then hybridized to the membrane-bound nucleic acid fragments. In addition, intact cDNA molecules may also be used, separated by electrophoresis, transferred to a membrane and analyzed by hybridization to labeled probes. Northern analyses, similarly, are conducted on nucleic acids, either intact or fragmented, that are bound to a membrane. The nucleic acids in Northern analyses, however, are generally RNA.  
           [0049]    3. Arrays  
           [0050]    Any microarray platform or technology maybe used to produce gene expression data that may be normalized with the control genes and methods of the invention. Oligonucleotide probe arrays can be made and used according to any techniques known in the art (see for example, Lockhart et al., (1996),  Nat Biotechnol  14: 1675-1680; McGall et al. (1996),  Proc Natl Acad Sci USA  93:13555-13460). Such probe arrays may contain at least one or more oligonucleotides that are complementary to or hybridize to one or more of the nucleic acids of the nucleic acid sample and/or the control genes of Table 1. Such arrays may also contain oligonucleotides that are complementary or hybridize to at least about 2, 3, 5, 7, 10, 25, 50, 100, 500 or more of the control genes listed in Table 1.  
           [0051]    Control oligonucleotide probes of the invention are preferably of sufficient length to specifically hybridize only to appropriate, complementary genes or transcripts. Typically the oligonucleotide probes will be at least about 10, 12, 14, 16, 18, 20 or 25 nucleotides in length. In some cases longer probes of at least 30, 40, or 50 nucleotides will be desirable. The oligonucleotide probes of high density array chips include oligonucleotides that range from about 5 to about 45, or 5 to about 500 nucleotides, more preferably from about 10 to about 40 nucleotides, and most preferably from about 15 to about 40 nucleotides in length. In other particularly preferred embodiments, the probes are 20 or 25 nucleotides in length. In another preferred embodiment, probes are double- or single-stranded DNA sequences. The oligonucleotide probes are capable of specifically hybridizing to the control gene nucleic acids in a sample.  
           [0052]    One of skill in the art will appreciate that an enormous number of array designs comprising control probes of the invention are suitable for the practice of this invention. The high density array will typically include a number of probes that specifically hybridize to each control gene nucleic acid, e.g. mRNA or cRNA (see WO 99/32660 for methods of producing probes for a given gene or genes). Assays and methods comprising control probes of the invention may utilize available formats to simultaneously screen at least about 100, preferably about 1000, more preferably about 10,000 and most preferably about 500,000 or 1,000,000 different nucleic acid hybridizations.  
           [0053]    The methods and control genes of this invention may also be used to normalize gene expression data produced using commercially available oligonucleotide arrays that contain or are modified to contain control gene probes of the invention. A preferred oligonucleotide array may be selected from the Affymetrix, Inc. GeneChipg series of arrays which include the Human Genome Focus Array, Human Genome U133 Set, Human Genome U95 Set, HuGeneFL Array, Human Cancer Array, HuSNP Mapping Array, GenFlex Tag Array, p53 Assay Array, CYP450 Assay Array, Rat Genome U34 Set, Rat Neurobiology U34 Array, Rat Toxicology U34 Array, Murine Genome U74v2, Murine 11K Set, Yeast Genome S98 Array,  E. coli  Antisense Genome Array,  E. coli  Genome Array (Sense), Arabidopsis ATH1 Genome Array, Arabidopsis Genome Array,  P. aeruginosa  Genome Array and  B. subtilis  Genome Array. In another embodiment, an oligonucleotide array may be selected from the Motorola Life Sciences and Amersham Pharmaceuticals CodeLink Bioarray System microarrays, including the UniSet Human 20K I, Uniset Human I, ADME-Rat, UniSet Rat I and UniSet Mouse I, or from the Motorola Life Sciences eSensor™ series of microarrays.  
           [0054]    4. RT-PCR  
           [0055]    The control genes and methods of the invention may be used in any type of polymerase chain reaction. A preferred PCR format is reverse transciptase polymerase chain reaction (RT-PCR), an in vitro method for enzymatically amplifying defined sequences of RNA (Rappolee et al. (1988),  Science  241: 708-712) permitting the analysis of different samples from as little as one cell in the same experiment (see “RT-PCR: The Basics,” Ambion, www.ambion.com/techlib/basics/rtpcr/index.html; PCR, M. J. McPherson and S. G. Moller, BIOS Scientific Publishers, Oxfordshire, England, 2000; and PCR Primer: A Laboratory Manual, Dieffenbach et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1995, for review). One of ordinary skill in the art may appreciate the enormous number of variations in RT-PCR platforms that are suitable for the practice of the invention, including complex variations aimed at increasing sensitivity such as semi-nested (Wasserman et al. (1999),  Mol Diag  4:21-28), nested (Israeli et al. (1994),  Cancer Res  54:6303-6310; Soeth et al. (1996),  Int J Cancer  69:278-282), and even three-step nested (Funaki et al. (1997),  Life Sci  60:643-652; Funaki et al. (1998),  Brit J Cancer  77:1327-1332).  
           [0056]    In one embodiment of the invention, separate enzymes are used for reverse transcription and PCR amplification Two commonly used reverse transcriptases, for example, are avian myeloblastosis virus and Moloney murine leukaemia virus. For amplification, a number of thermostable DNA-dependent DNA polymerases are currently available, although they differ in processivity, fidelity, thermal stability and ability to read modified triphosphates such as deoxyuridine and deoxyinosine in the template strand (Adams et al. (1994),  Bioorg Med Chem  2:659-667; Perler et al. (1996),  Adv Prot Chem  48:377-435). The most commonly used enzyme, Taq DNA polymerase, has a 5′-3′ nuclease activity but lacks a 3′-5′ proofreading exonuclease activity. When fidelity is required, proofreading exonucleases such as Vent and Deep Vent (New England Biolabs) or Pfu (Stratagene) may be used (Cline et al. (1996),  Nucl Acids Res  24:3456-3551). In another embodiment of the invention, a single enzyme approach maybe used involving a DNA polymerase with intrinsic reverse transcriptase activity, such as  Thermus thermophilus  (Tth) polymerase (Bustin (2000),  J Mol Endo  25:169-193). A skilled artisan may appreciate the variety of enzymes available for use in the present invention.  
           [0057]    The methodologies and control gene primers of the present invention may be used, for example, in any kinetic RT-PCR methodology, including those that combine fluorescence techniques with instrumentation capable of combining amplification, detection and quantification (Orlando et al. (1998),  Clin Chem Lab Med  36:255-269). The choice of instrumentation is particularly important in multiplex RT-PCR, wherein multiple primer sets are used to amplify multiple specific targets simultaneously. This requires simultaneous detection of multiple fluorescent dyes. Accurate quantitation while maintaining a broad dynamic range of sensitivity across mRNA levels is the focus of upcoming technologies, any of which are applicable for use in the present invention. Preferred instrumentation may be selected from the ABI Prism 7700 (Perkin-Elmer Applied Biosystems), the Lightcycler (Roche Molecular Biochemicals) and icycler Thermal Cycler. Featured aspects of these products include high-throughput capacities or unique photodetection devices.  
           [0058]    Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, practice the methods and use the control genes of the present invention. The following examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. 
       
    
    
     EXAMPLES  
     Example 1  
     Selection of Control Genes  
       [0059]    The control genes were selected by querying a Gene Logic rat tissue database to create expression profiles from a variety of rat cell and tissue samples.  
         [0060]    This database was produced from data derived from screening various cell or tissue samples using an Affymetrix rat GeneChip® set. The rat cell and tissue samples that were analyzed include those that were not treated at all and that can be referred to as “normal,” as they represent the laboratory rat population that has not been manipulated outside of normal daily activity within that setting. In general, tissue and cell samples were processed following the Affymetrix GeneChip® Expression Analysis Manual. Frozen tissue or cells were ground to a powder using a Spex Certiprep 6800 Freezer Mill. Total RNA was extracted with Trizol (GibcoBRL), according to the manufacturer&#39;s protocol. The total RNA yield for each sample was 200-500 μg per 300 mg cells. mRNA was isolated using the Oligotex mRNA Midi kit (Qiagen) followed by ethanol precipitation. Double stranded cDNA was generated from mRNA using the SuperScript Choice system (GibcoBRL). First strand cDNA synthesis was primed with a T7-(dT24) oligonucleotide. The cDNA was phenol-chloroform extracted and ethanol precipitated to a final concentration of 1 μg/ml. From 2 μg of cDNA, cRNA was synthesized using Ambion&#39;s T7 MegaScript in vitro Transcription Kit.  
         [0061]    To biotin label the cRNA, nucleotides Bio-11-CTP and Bio-16-UTP (Enzo Diagnostics) were added to the reaction. Following a 37° C. incubation for six hours, impurities were removed from the labeled cRNA following the RNeasy Mini kit protocol (Qiagen). cRNA was fragmented (fragmentation buffer consisting of 200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc) for thirty-five minutes at 94° C. Following the Affymetrix protocol, 55 μg of fragmented cRNA was hybridized on an Affymetrix Rat Genome U34 array set for twenty-four hours at 60 rpm in a 45° C. hybridization oven. The chips were washed and stained with Streptavidin Phycoerythrin (SAPE) (Molecular Probes) in Affymetrix fluidics stations. To amplify staining, SAPE solution was added twice with an anti-streptavidin biotinylated antibody (Vector Laboratories) staining step in between. Hybridization to the probe arrays was detected by fluorometric scanning (Hewlett Packard Gene Array Scanner). Following hybridization and scanning, the chips were analyzed for quality control, looking for major chip defects or abnormalities in hybridization signal. After the chips passed quality control, data were analyzed using Affymetrix GeneChip® version 3.0 and Expression Data Mining Tool (EDMT) software (version 1.0), S-Plus, and the GeneExpresss software system. Microarrays were scanned on a high photomultiplier tube (PMT) settings.  
         [0062]    To prepare tissue samples from animals, e.g., rats, sterile instruments were used to sacrifice the animals, and fresh and sterile disposable instruments were used to collect tissues. Gloves were worn at all times when handling tissues or vials. All tissues were collected and frozen within approximately 5 minutes of the animal&#39;s death. The liver sections and kidneys were frozen within approximately 3-5 minutes of the animal&#39;s death. The time of euthanasia, an interim time point at freezing of liver sections and kidneys, and time at completion of necropsy were recorded. Tissues were stored at approximately −80° C. or perserved in 10% neutral buffered formalin. Tissues were collected and processed as follows.  
         [0063]    Liver  
         [0064]    1. Right medial lobe—snap frozen in liquid nitrogen and stored at −80° C.  
         [0065]    2. Left medial lobe—Preserved in 10% neutral-buffered formalin (NBF) and evaluated for gross and microscopic pathology.  
         [0066]    3. Left lateral lobe—snap frozen in liquid nitrogen and stored at −80° C.  
         [0067]    Heart—A sagittal cross-section containing portions of the two atria and of the two ventricles was preserved in 10% NBF. The remaining heart was frozen in liquid nitrogen and stored at −80° C.  
         [0068]    Kidneys (both)  
         [0069]    1. Left—Hemi-dissected; half was preserved in 10% NBF and the remaining half was frozen liquid nitrogen and stored at −80° C.  
         [0070]    2. Right—Hemi-dissected; half was preserved in 10% NBF and the remaining half frozen in liquid nitrogen and stored at −80° C.  
         [0071]    Testes (both)—A sagittal cross-section of each testis was preserved in 10% NBF. The remaining testes were frozen together in liquid nitrogen and stored at −80° C.  
         [0072]    Brain (whole)—A cross-section of the cerebral hemispheres and of the diencephalon was preserved in 10% NBF, and the rest of the brain was frozen in liquid nitrogen and stored at −80° C.  
         [0073]    Gene expression data were then analyzed to identify those genes that were consistently expressed across a set of about 5,000 different tissue samples, e.g., being called Present more than 95% of the time. For each of these samples, the mean average difference, standard deviation and CV were determined for each Affymetrix fragment on the rat U34 GeneChip®. The data were sorted by CV, and those gene fragments with values less than 40% were chosen for further analysis. Table 1 provides a list of approximately 858 genes with a coefficient of variation less than 0.44 and whose expression is considered not to vary across the normal and treated samples studied. For each gene listed, Table 1 also provides a GenBank Accession No., a Present frequency value, a mean expression level value and a coefficient of variation, expressed as CV. The GenBank Accession Nos. can be used to locate the publicly available sequences, each of which is herein incorporated by reference in its entirety as of the priority date of this application (Jul. 30, 2002).  
       Example 2  
     Quantitative PCR Analysis of Expression Levels Using the Control Genes  
       [0074]    The expression levels of one or more genes listed in Table 1 may be used to normalize gene expression data produced using quantitative PCR analysis. For example, the sequences may be used as Taqman® probes, along with the forward and reverse primers for a gene in Table 1. Real time PCR detection may be accomplished by the use of the ABI PRISM 7700 Sequence Detection System. The 7700 measures the fluorescence intensity of the sample each cycle and is able to detect the presence of specific amplicons within the PCR reaction. The TaqMan® assay provided by Perkin Elmer may be used to assay quantities of RNA. The primers may be designed from each of the genes identified in Table 1 using Primer Express, a program developed by PE to efficiently find primers and probes for specific sequences. These primers may be used in conjunction with SYBR green (Molecular Probes), a nonspecific double-stranded DNA dye, to measure the expression level of mRNA corresponding to the expression levels of each gene. This gene expression data may then be used to normalize gene expression data of other test genes.  
         [0075]    Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All cited patents and publications referred to in this application are herein incorporated by reference in their entirety.  
                                                         TABLE 1                                       Present                   GenBank No.   Frequency   Adjusted Mean   CV                                        NM_057141   0.9621   353.7302949   0.394573166           AA800364   0.9921   538.7477202   0.35144586            AA800501   0.9874   200.464431   0.271392863           AA801051   0.9946   594.9934288   0.327732429           AA801442   0.9937   472.4598982   0.314834757           AA848238   0.9516   341.660987   0.288832086           AA849262   0.9846   210.520306   0.37721446            AA944127   0.9522   178.105728   0.384011401           NM_031981   0.994    328.1689155   0.389666675           NM_031981   0.998    384.2180916   0.31917883            NM_019352   0.9964   577.4245502   0.233685612           AB008807   0.9906   375.3583161   0.389113636           NM_019213   0.9959   167.7105557   0.33117565            NM_031331   0.9928   299.5668687   0.388714827           NM_019191   0.9725   67.90177915   0.330844052           NM_053527   0.9608   151.0502046   0.316326745           AF003926   0.9947   331.151405   0.243708921           NM_031656   0.9624   70.8858116   0.349709856           NM_053553   0.9656   136.6785634   0.375165396           NM_053556   0.9816   193.2494812   0.365428046           NM_019201   0.9993   800.7665129   0.383779573           NM_022536   0.992    637.6998997   0.349019258           NM_031749   0.9833   260.969288   0.379520142           AF093139   0.9955   164.5811247   0.291070695           NM_053467   0.9967   645.032758   0.312688813           NM_019222   0.9959   241.1715455   0.306923163           NM_053707   0.9954   278.2968887   0.363812568           NM_057143   0.9969   491.5701144   0.377464693           NM_057141   0.99    334.7099186   0.372595428           NM_017284   0.9991   469.0115461   0.37489808            NM_053743   0.9986   599.9253754   0.318539549           NM_031603   0.9961   531.1713873   0.394886131           037934   0.9794   212.2995269   0.267710366           NM_022598   0.9898   150.3146804   0.392695104           NM_022598   0.9978   410.4386698   0.355948912           NM_013076   0.0736   249.8757424   0.364628324           NM_019317   0.9734   83.51973777   0.373249306           NM_017236   0.9867   671.5891964   0.360897244           H32978   0.997    435.2398828   0.300833768           NM_031090   0.9619   70.31528575   0.399983405           NM_057209   0.9864   257.5044748   0.332226154           NM_012500   0.9895   150.6522809   0.302162035           K02816   0.9981   382.6421388   0.334260892           NM_022518   0.99    575.2287493   0.267122194           NM_031129   0.9986   672.686873   0.268407165           NM_012639   0.9592   157.9459425   0.307400434           NM_031974   0.9978   616.4278739   0.361748118           NM_013177   0.9988   787.1641147   0.381937146           NM_017101   0.9975   1067.896541   0.347227639           M57728   0.9729   108.3973358   0.368600327           AA684641   0.9692   132.9106769   0.312063584           AA799279   0.9991   839.3057142   0.325266583           AA799279   0.9947   568.1583462   0.347844769           AA799542   0.9924   273.5836187   0.370208871           AA799550   0.9973   470.9384047   0.370333288           AA799609   0.9912   134.1295318   0.334268614           AA799641   0.9966   276.4144125   0.307718893           AA799654   0.9981   296.1941725   0.351166278           AA799667   0.9908   248.9277967   0.291789627           AA799721   0.9629   114.4838534   0.373755794           AA799735   0.9644   126.8477716   0.292430382           AA799735   0.9813   137.5032487   0.318140687           AA799822   0.9906   162.7568631   0.360262563           NM_033096   0.9941   225.0546461   0.319505767           AA800015   0.9972   384.3536135   0.289608893           AA800039   0.9906   354.1901013   0.287620068           AA800053   0.9898   129.5213675   0.37530915            AA800170   0.9675   75.9629053   0.355273922           AA800198   0.9639   159.2105578   0.295644976           AA800210   0.9821   105.0330379   0.370992795           NM_013006   0.9898   237.3041636   0.391423327           AA800268   0.976    166.4768623   0.340868372           AA800651   0.9912   400.5374777   0.330167434           AA800669   0.9949   426.0164527   0.393889597           AA800787   0.9874   149.0104015   0.379600998           AA800814   0.9525   109.058537   0.389571008           AA801130   0.9957   263.9245532   0.38007823            AA801176   0.989    325.5564512   0.295890207           AA801230   0.9972   567.3071148   0.389999402           NM_032057   0.9955   179.3952918   0.296133732           AA817769   0.9941   185.0590031   0.323946319           AA817845   0.9951   380.1019029   0.242937824           NM_053682   0.9941   267.5028747   0.372604104           AA817892   0.9828   305.8321361   0.370745167           AA817907   0.9916   285.4664154   0.323851183           AA817945   0.9979   1077.847309   0.363411979           AA817967   0.9943   296.89826   0.323986488           AA818118   0.9951   324.9041145   0.349051053           PA818129   0.99    187.622771   0.301614267           NM_130405   0.9909   169.6695017   0.325635921           AA818203   0.9931   173.3889032   0.372430825           AA818246   0.9878   423.0700233   0.395146083           AA818534   0.9927   259.4059044   0.283803337           AA818568   0.9772   79.08353703   0.299639563           AA818669   0.9928   330.9312721   0.317467573           AA818697   0.9979   645.1875312   0.274054292           AA818778   0.9964   324.6127355   0.343055107           AA818788   0.988    128.4093671   0.374028119           NM_019907   0.993    178.4123338   0.361138088           AA819057   0.9974   559.6063582   0.246931544           AA819119   0.9621   91.25127707   0.380140187           AA819224   0.9812   135.2326929   0.383447682           NM_031745   0.9853   165.1417753   0.394406392           AA819318   0.9527   212.4831719   0.361885928           AA819362   0.9862   154.0922099   0.361381365           AA819364   0.9933   282.8186095   0.260938603           AA819367   0.991    135.7415399   0.34405043            AA819400   0.9886   135.3139207   0.345055159           AA819468   0.9986   320.3062492   0.334844865           AA819471   0.9678   102.5559907   0.342984629           AA819487   0.9736   138.4349905   0.345244474           AA819691   0.9941   431.8944648   0.382341107           AA819694   0.9714   103.4099525   0.372870205           AA819729   0.9931   289.9616028   0.32509288            AA819761   0.987    240.328863   0.353298909           AA819798   0.9961   412.8591164   0.38513809            AA819798   0.9977   543.3893727   0.333884343           AA848404   0.9812   470.973766   0.353247324           NM_133320   0.9901   374.0195578   0.370573918           AA848674   0.9709   198.5709918   0.268430905           AA848696   0.9584   113.9625472   0.393992922           AA848967   0.9551   371.1002902   0.378302744           AA849092   0.9941   265.2055344   0.361330207           AA849312   0.9896   261.6560729   0.302612314           AA849531   0.9977   452.3976272   0.258409286           AA849715   0.9955   427.4012934   0.324344933           AA849721   0.9759   429.6255416   0.3397144            AA849757   0.991    728.7550103   0.385766928           AA849766   0.9905   338.2837276   0.386484508           AA849767   0.9892   686.473722   0.370408924           AA849774   0.9543   199.0178366   0.290368511           AA849788   0.9899   277.0979159   0.302937581           AA849809   0.9847   213.0699948   0.395797677           AA849952   0.9836   249.6829894   0.317677787           AA849954   0.9523   86.97070267   0.337457286           AA849965   0.9908   209.8670776   0.346431239           AA850117   0.9611   228.5306101   0.38524339            AA850451   0.9922   427.5517004   0.379543087           AA850480   0.9932   408.6763631   0.386935606           AA850525   0.9702   335.2964034   0.260647679           AA850529   0.9771   383.9424729   0.372557101           AA850535   0.994    484.8463265   0.306355053           AA850550   0.9955   538.4247695   0.220155894           AA850569   0.9969   539.4661192   0.369801777           AA850624   0.966    114.0740815   0.388810835           AA850666   0.9933   372.2546801   0.294655078           AA850754   0.9731   100.307608   0.376838798           AA850894   0.9917   442.4665358   0.273072964           AA850907   0.9935   365.9563743   0.326461416           AA851161   0.9968   448.0118551   0.319626206           AA851202   0.9808   200.0256289   0.355469087           AA851214   0.9914   523.3549456   0.295434486           AA851251   0.996    296.8962387   0.303746             AA851347   0.9982   438.4096815   0.299495219           AA851376   0.9982   499.552633   0.311653073           AA851397   0.9651   325.960527   0.365291903           AA851405   0.9773   114.6840333   0.327907652           AA851439   0.962    229.2705115   0.326726191           AA851464   0.9918   267.8901625   0.284156685           AA851641   0.979    179.8467866   0.376196046           NM_133324   0.9766   178.8689584   0.271247953           AA851686   0.9954   300.5452993   0.237099425           AA851701   0.9737   134.3344569   0.344149947           AA851728   0.9785   252.4667912   0.323521957           AA851739   0.9553   206.4328905   0.300390557           AA851765   0.97    828.6859018   0.305562042           AA851873   0.9721   329.8649568   0.359666828           AA851883   0.977    185.2628058   0.329071104           AA851909   0.9906   206.1724423   0.31357168            AA851920   0.9779   208.7672739   0.374968241           AA851938   0.9938   392.3178541   0.326446676           AA858457   0.9843   168.426778   0.309690174           NM_031153   0.9815   289.8076299   0.3760569            AA858551   0.9893   210.2360092   0.362121119           AA858660   0.9962   252.2957635   0.34707555            AA858718   0.9977   1028.984349   0.399132237           AA858833   0.9793   159.8825966   0.393184202           AA858867   0.9648   136.1726528   0.331092316           AA858990   0.9965   870.3627949   0.365647982           AA859100   0.9772   136.2773269   0.356567976           AA859201   0.9978   275.683128   0.306043339           AA859796   0.9534   71.01143176   0.389965008           AA859919   0.9881   142.7674515   0.333076793           AA859919   0.9988   667.1537331   0.292027993           AA866364   0.9931   138.1245174   0.38210287            AA866371   0.9633   142.6912204   0.320447204           NM_024394   0.9617   151.4019916   0.387696691           AA875431   0.9929   254.161646   0.360714718           AA875470   0.9605   291.3569793   0.273123402           AA875470   0.9683   127.7332542   0.328086924           AA875552   0.9933   196.7545027   0.275253333           AA875661   0.9853   74.47796632   0.335756096           NM_053739   0.9971   223.2643235   0.343236121           AA891546   0.972    65.30502026   0.399473194           AA891717   0.9819   135.7970398   0.291717192           AA891742   0.9844   120.1613409   0.361978153           AA891746   0.9946   310.8827948   0.393391849           AA891810   0.9808   328.0727833   0.30475391            AA891810   0.9858   152.5190759   0.339434397           AA891902   0.9702   51.59142223   0.349786011           AA891935   0.988    233.0024719   0.270449187           AA892120   0.9791   60.97800731   0.373214916           AA892313   0.9913   107.2159627   0.389027092           AA892394   0.9971   221.3345267   0.386151078           AA892394   0.9952   129.1994687   0.397846098           AA892422   0.975    185.0194316   0.252043297           AA892505   0.9941   256.1674794   0.272080589           AA892550   0.9702   118.6361973   0.377480727           AA892789   0.986    236.3026727   0.297799259           AA892791   0.9855   175.6311243   0.325854158           AA892796   0.9973   621.5110927   0.313521756           AA892814   0.9959   421.7165288   0.32340475            AA893224   0.9918   129.2966135   0.319716751           AA893353   0.9939   289.2938276   0.343012426           AA893515   0.9965   268.1693134   0.293905313           AA893641   0.9655   124.631513   0.343387848           AA893683   0.983    87.44882196   0.347534606           AA893741   0.9921   192.2533724   0.269508199           AA893811   0.9736   84.60301216   0.398222929           AA894099   0.9813   285.142634   0.328324892           AA894101   0.9559   102.4471478   0.332265391           AA894101   0.9824   111.4560965   0.350773318           AA894131   0.9766   106.387669   0.383457001           AA894234   0.9841   236.1264994   0.284552291           AA894259   0.9966   449.0454763   0.312420033           AA899546   0.982    220.5971506   0.309976207           AA899672   0.9979   1656.075895   0.319960146           AA899691   0.9924   195.4072733   0.34078252            AA899743   0.9974   1071.973786   0.330354696           AA899911   0.9976   522.9454135   0.252669377           AA899959   0.9952   492.0446142   0.395266737           AA900078   0.9565   188.0606334   0.387115384           AA900156   0.9857   761.5282734   0.223294586           AA900187   0.998    471.5301948   0.257859064           AA900343   0.9773   209.1604636   0.322304252           AA900348   0.9502   212.7503091   0.340891055           AA900364   0.9652   162.5703442   0.284958346           AA900422   0.9604   404.2714995   0.356451649           AA900860   0.9869   167.3921533   0.383802956           AA900891   0.9524   149.0980669   0.315160786           AA900975   0.9978   868.1886108   0.37200699            AA901222   0.9956   480.1616103   0.309579403           AA901365   0.9995   890.2613004   0.32691327            AA923992   0.9576   114.2571622   0.396496378           AA923998   0.9659   225.9503235   0.280597467           AA924030   0.989    162.9127727   0.392729941           AA924079   0.9821   205.1877284   0.379629317           AA924092   0.9888   318.9907396   0.228510957           AA924169   0.9927   288.0750562   0.272550639           AA924317   0.9867   190.5012975   0.224003653           AA924339   0.999    1652.670033   0.282834255           AA924369   0.9936   365.6561614   0.261149514           AA924532   0.9632   344.493183   0.317913577           NM_031020   0.9604   62.91020969   0.397017329           AA924604   0.9938   318.2624476   0.354029504           AA924609   0.9984   461.8011067   0.378571404           AA924654   0.9809   211.2829082   0.332834334           NM_053555   0.9507   311.2721659   0.318866213           AA924765   0.9972   324.8928559   0.224049302           AA924768   0.9896   599.7543673   0.309926225           AA924787   0.9896   481.0525157   0.333960186           AA924871   0.9647   148.8977646   0.393740956           AA925123   0.9984   850.7664442   0.264758138           AA925152   0.9946   699.0355291   0.239167455           AA925160   0.9823   190.1916631   0.373116063           AA925212   0.9973   611.8057286   0.294395182           AA925304   0.9902   302.2840247   0.272196777           AA925305   0.9837   411.7095514   0.285568444           AA925338   0.9803   298.8774464   0.290626881           AA925340   0.9991   521.8490052   0.283743847           AA925341   0.9669   241.6436392   0.360237623           AA925432   0.9735   225.7988151   0.350901777           AA925473   0.9959   622.4378044   0.399479916           AA925478   0.9819   341.7412759   0.372375386           AA925677   0.9608   201.0099572   0.335202855           AA925854   0.9842   201.3893423   0.31172844            AA925979   0.9878   458.165257   0.346902976           AA925983   0.999    599.5384168   0.391862852           AA926013   0.981    193.6558006   0.228102661           AA926098   0.9965   755.140021   0.247890637           AA926279   0.9703   258.1851509   0.330058893           AA926331   0.9658   142.9219708   0.389925982           AA933158   0.9771   163.6147359   0.267516634           AA942947   0.9693   175.6990963   0.375870721           AA943015   0.9726   170.0338035   0.360085756           AA943015   0.9858   388.2429204   0.341776907           AA943122   0.9762   419.8206702   0.320372184           AA943240   0.9636   203.3959179   0.34869085            AA943281   0.9691   305.0288964   0.378822839           NM_012913   0.9813   258.9236246   0.388849176           AA943421   0.9632   209.3195009   0.370589173           AA943500   0.9708   209.919368   0.281165988           AA943553   0.9872   519.9751425   0.382535341           AA943553   0.9966   665.5611215   0.379984839           AA943645   0.9933   581.0411338   0.381146596           AA943738   0.9859   137.0917646   0.271120535           AA943766   0.9957   400.0460991   0.370001453           NM_080909   0.9928   872.5315577   0.389923883           AA944203   0.9914   400.6244567   0.350853364           AA944335   0.9976   866.0289208   0.267386275           AA944347   0.9626   161.3067747   0.318180188           AA944445   0.9781   222.5978106   0.373397302           AA944451   0.9971   599.870325   0.323030108           AA944528   0.9954   425.8651494   0.22460926            AA944635   0.9801   322.8449619   0.312266733           AA944842   0.9861   299.6522749   0.237903089           AA945089   0.9941   914.5995769   0.375028263           NM_133297   0.994    683.5483047   0.367321409           AA945740   0.9612   124.6495711   0.329660581           AA945746   0.9956   408.4484576   0.33612417            AA945746   0.9839   255.5524434   0.348157071           NM_131907   0.9962   370.0878264   0.339124904           AA945869   0.9792   179.2868431   0.327315212           AA946004   0.983    142.7065978   0.295987651           AA946018   0.9978   786.5592317   0.233292925           AA946038   0.9888   281.4038976   0.278216507           AA946205   0.9976   828.2592325   0.35788087            AA946432   0.9924   545.660241   0.259340094           AA946440   0.9982   570.5688594   0.245347075           AA955112   0.9962   291.1739164   0.212582952           AA955240   0.9973   647.7129713   0.274411246           NM_017359   0.996    231.4699675   0.329048264           AA955396   0.9879   243.1046885   0.255401546           AA955506   0.9877   242.1871379   0.383670644           AA955536   0.9823   187.9185985   0.269361535           AA956114   0.9955   143.3842105   0.398204182           AA956140   0.9938   776.6322184   0.395628638           AA956185   0.9823   250.4214737   0.341205084           AA956460   0.9955   399.3603811   0.336899504           AA956983   0.9853   295.1596861   0.34098573            AA956992   0.9928   417.4006281   0.269624667           AA956992   0.9976   491.7470975   0.243689773           AA957063   0.9941   391.7747852   0.319424296           AA957491   0.988    180.2576966   0.351832394           AA957649   0.9924   423.4676789   0.338824147           AA957676   0.9592   429.4318847   0.396293319           AA957777   0.9866   112.7434987   0.363054879           AA963072   0.9709   134.5270945   0.333884657           AA963094   0.9977   606.8333854   0.328724125           AA963170   0.987    118.5722127   0.275144443           AA963367   0.9977   817.7237717   0.369667036           AA963808   0.998    669.6077262   0.382254088           AA964054   0.9949   405.6577401   0.347892099           AA964064   0.9888   375.2686433   0.37526759            AA964082   0.9956   680.4697645   0.272604335           AA964114   0.9831   753.7315494   0.29001828            AA964362   0.9869   145.3535334   0.33101453            AA964366   0.9774   316.6869935   0.283982561           AA964607   0.9923   499.9287489   0.320740471           AA964624   0.9538   125.8056987   0.280619534           AA964630   0.993    389.0162941   0.283601586           AA964642   0.9755   372.5717109   0.358580387           AA965073   0.9802   626.0264683   0.341947106           AA996398   0.9501   142.9854577   0.356277705           AA996576   0.9856   411.2372292   0.313642878           AA996797   0.9889   366.872934   0.310438476           AA996939   0.994    369.2356692   0.339669675           AA996974   0.9859   172.2669572   0.330914903           AA997052   0.9625   159.3897659   0.386386551           AA997184   0.9801   277.3537497   0.296759505           NM_053494   0.982    300.8210503   0.361139323           AA997929   0.9888   166.2636207   0.279152327           AA998158   0.9521   297.1376512   0.280727643           AA998435   0.9989   878.2922689   0.231761767           AA998523   0.9678   182.5520659   0.383305234           AA998556   0.9802   157.5036188   0.323268276           AA998843   0.9713   200.1415524   0.351313361           AA998893   0.9938   218.6451718   0.356607867           AI007743   0.9885   280.3187898   0.397692815           AI007750   0.9766   224.5812217   0.29763571            AI007920   0.9603   128.7892564   0.362125165           AI007987   0.9945   391.0597095   0.286018393           AI008372   0.996    586.5742499   0.29223874            AI008423   0.9933   197.7269351   0.251485242           AI008683   0.9969   590.7294525   0.250069145           AI008740   0.9606   182.2744427   0.352124819           AI008774   0.995    226.9116964   0.278990202           AI008784   0.9968   1245.190937   0.28202077            AI008931   0.9798   214.3402379   0.369067812           AI008958   0.9956   1352.486047   0.283940464           AI009079   0.9974   826.1352041   0.383871133           AI009157   0.9919   213.2822268   0.364389086           AI009200   0.9967   440.7137578   0.313553376           AI009350   0.9998   711.4169929   0.316543066           NM_053416   0.9988   2751.842839   0.354727984           AI009591   0.9601   115.0663701   0.297851506           AI009650   0.9861   290.3570616   0.313940175           AI009655   0.9884   408.8257028   0.386816933           AI009693   0.9945   704.5208126   0.38652766            AI009741   0.9983   502.0441942   0.347210566           AI009772   0.9982   661.4997157   0.357510104           AI009819   0.9833   385.974512   0.30650183            AI009936   0.9982   482.9852102   0.382801193           AI010034   0.9869   175.1325883   0.357119903           AI010342   0.9757   160.9745004   0.341899927           AI010362   0.9879   325.1416967   0.367946604           AI010422   0.9766   133.9096768   0.348370485           AI010452   0.9995   1364.399147   0.323158828           AI010518   0.9897   523.3835278   0.251025801           AI010758   0.9791   159.5785487   0.342965544           AI010944   0.9655   198.931889   0.348891534           AI011148   0.9888   509.7577822   0.297487627           AI011190   0.9848   244.2595909   0.263968956           AI011306   0.9735   186.9337855   0.299628693           AI011339   0.9878   339.3332871   0.295719531           AI011344   0.9985   808.9708186   0.242918026           AI011556   0.9933   307.1623657   0.361656015           AI011571   0.9913   231.1039866   0.349951837           AI011754   0.9601   357.2305561   0.259066046           AI011756   0.9845   505.6862363   0.335393314           AI012027   0.9905   498.9415909   0.330533351           AI012077   0.9685   183.5304607   0.28972612            AI012258   0.9627   232.9319168   0.397616077           AI012277   0.9815   224.0156956   0.315069042           AI012285   0.9969   455.2112947   0.251368311           AI012467   0.9755   247.9979552   0.393536674           AI012562   0.9819   247.0055648   0.399176309           AI012567   0.994    614.0183082   0.300419775           AI012636   0.9774   244.7954881   0.343753177           AI012641   0.9954   1107.58001   0.232410874           AI012937   0.9873   231.1042578   0.299231915           AI012947   0.9972   750.634375   0.34167501            AI012951   0.9969   589.2139983   0.348304745           AI013024   0.9941   972.6981993   0.377775478           AI013090   0.9848   294.8100632   0.307498244           AI013097   0.9949   470.59474   0.279198054           AI013204   0.9984   974.7028181   0.387451663           AI013350   0.9844   259.9148955   0.232030679           AI013363   0.9974   517.4391968   0.277623342           AI013555   0.9             335.5652187   0.285524016           AI013564   0.9686   181.8970045   0.347588318           AI013697   0.9987   911.7514272   0.330567711           NM_031721   0.9825   322.6669671   0.384108704           AI013816   0.9654   635.7636797   0.31640618            AI013870   0.9903   267.8569007   0.324199211           AI013946   0.9985   532.8561833   0.216157143           AI014059   0.9806   391.8286644   0.306162721           AI028997   0.9851   246.3910601   0.293805901           AI029110   0.9941   292.5132162   0.268267155           AI029421   0.9787   293.3496299   0.204319564           AI029484   0.9977   367.1680693   0.227864083           AI029733   0.9987   1950.541312   0.296475897           AI029737   0.9916   520.438801   0.25582686            AI030147   0.9894   307.9855538   0.350690338           AI030192   0.9753   234.1326385   0.387161574           AI030248   0.9969   701.8459509   0.250558161           AI030430   0.9955   511.3544152   0.329914711           AI030751   0.9565   233.3235568   0.269114645           AI030799   0.9962   490.648901   0.386351868           AI030907   0.9939   322.801128   0.367771191           AI031035   0.9978   352.4611173   0.295253508           AI044112   0.9956   355.4596171   0.308803476           NM_053864   0.9816   329.9527469   0.325582005           AI044727   0.9941   399.1541549   0.24747723            NM_022595   0.9749   558.6476666   0.376778078           AI044863   0.9792   231.3400497   0.328631874           AI044872   0.9738   126.3552963   0.319073417           AI045003   0.9781   302.1675806   0.367003326           NM_053347   0.9792   425.6707106   0.262323344           AI045458   0.9987   834.2478872   0.281073528           AI045597   0.9795   236.7872339   0.362316181           AI045774   0.9984   597.5232402   0.361381584           Al045810   0.9758   119.8442776   0.37446961            AI058373   0.9714   166.5305651   0.310968224           AI058963   0.9795   226.9015804   0.318390827           AI058972   0.9639   163.4753435   0.314357814           AI059428   0.9631   337.5326409   0.343346037           AI059762   0.9628   101.8533271   0.350501189           AI060132   0.9923   869.1205218   0.296469343           AI060196   0.9987   652.5830242   0.247507825           AI060222   0.9954   274.2012257   0.299764113           AI070070   0.9762   237.8818411   0.32422811            AI070153   0.9524   241.7017398   0.274003857           AI070176   0.9983   508.310541   0.347627491           AI070399   0.9921   240.2738781   0.320172095           AI071243   0.9775   164.5641109   0.375047226           AI071773   0.9948   176.2687515   0.362438965           AI071946   0.9964   224.3185087   0.299033252           AI072081   0.9983   581.6999055   0.319003258           AI072121   0.9872   250.8923104   0.328781873           AI072555   0.9637   103.7773054   0.269538712           AI072666   0.9947   470.2436446   0.322438521           AI072675   0.9957   324.3957217   0.378467368           AI072885   0.9812   128.3180767   0.328993951           AI073030   0.997    529.5599398   0.226307567           AI073118   0.9816   132.8195789   0.330656304           AI073193   0.9983   387.0532434   0.314540172           AI073215   0.9879   422.9032397   0.327263785           AI073260   0.999    990.0658894   0.383685551           NM_053569   0.9896   203.3512567   0.226791767           AI101181   0.9741   97.17989413   0.28235925            AI101222   0.993    312.149685   0.31926142            AI101375   0.9933   415.4787877   0.391617213           AI101395   0.9725   160.7363746   0.388039479           AI101438   0.9709   64.45239629   0.38983282            AI101460   0.9974   399.5143745   0.355650986           AI101659   0.9988   627.0523045   0.329943             AI101864   0.9983   1112.010461   0.276584797           AI101934   0.973    160.6040766   0.331698393           AI102046   0.9785   187.0808649   0.28794593            AI102080   0.9882   291.239455   0.268077727           AI102191   0.9768   160.2239891   0.276862462           AI102252   0.9936   186.9971017   0.289805898           NM_053436   0.973    227.2297147   0.385866717           AI102438   0.9956   321.3897237   0.394869448           AI102612   0.9975   571.3800141   0.289213412           AI102734   0.9938   530.6640201   0.385875553           AI102935   0.9884   324.8164874   0.3850101            AI102978   0.9903   147.7101205   0.381554081           AI102991   0.998    389.6494934   0.211087424           AI103094   0.9979   873.9486249   0.282128391           AI103129   0.9774   740.612351   0.322658411           NM_031146   0.9852   438.0155262   0.279684593           AI103377   0.9844   226.9637436   0.305656574           AI103379   0.9981   528.362849   0.191923868           AI103428   0.9832   141.4527273   0.375933874           AI103521   0.9907   384.7084269   0.310270528           AI103717   0.95    277.674925   0.301083424           AI103718   0.998    991.3056425   0.220129724           AI103848   0.9836   146.2910591   0.304856826           AI103950   0.9684   85.7813618   0.38997044            AI103954   0.9965   345.2301178   0.370824066           AI104231   0.9752   132.4494652   0.325845881           AI104234   0.9786   374.7482362   0.356449801           AI104239   0.979    125.3204702   0.332461387           AI104247   0.9691   212.5064712   0.340422775           AI104250   0.9641   219.0354755   0.325898084           AI104283   0.9922   289.7160716   0.266096751           AI104320   0.9906   303.2373949   0.248088041           AI104388   0.9505   156.6673508   0.329438846           AI104488   0.9672   117.8088465   0.229489312           AI104536   0.9986   1025.536272   0.284465579           NM_022518   0.9936   711.8999891   0.304763664           AI104600   0.9521   122.2863459   0.349242431           AI104753   0.9524   326.7032346   0.399036819           AI104864   0.9868   376.8258789   0.356488841           AI104878   0.9972   438.505944   0.358693351           AI104914   0.9956   199.5044251   0.268591505           NM_080781   0.9663   107.7513281   0.335662126           AI105072   0.9972   395.9783073   0.387243663           NM_057205   0.9978   283.3131956   0.293441255           AI105087   0.9933   515.1104067   0.352504039           AI105149   0.9983   911.5392665   0.263156658           AI105265   0.9538   217.2837741   0.341799777           AI105345   0.9861   155.8460745   0.364213518           AI105352   0.9938   141.9075167   0.361489718           AI105431   0.998    356.6841375   0.378020827           AI111683   0.9915   184.2053628   0.241117848           AI111975   0.999    192.3560148   0.3351647            AI112092   0.9954   269.3073934   0.349380313           AI112250   0.9968   653.2935303   0.378593708           AI112512   0.9598   75.40270266   0.386823108           AI113020   0.9844   232.0838805   0.311528728           AI136231   0.9537   132.4605103   0.376730451           AI136564   0.9761   278.6318378   0.35005281            AI136669   0.9958   518.5194087   0.351974463           AI137232   0.9988   469.6158446   0.366380187           AI137298   0.9923   230.6509496   0.270580577           AI137582   0.9799   135.3498294   0.397497765           AI138002   0.9942   219.6829104   0.24859447            AI144657   0.9923   129.4479951   0.312677319           AI144668   0.9909   297.1273683   0.314893774           AI144956   0.9904   207.7546316   0.264521312           AI145332   0.9538   129.0242513   0.37788731            AI145362   0.9719   120.3802137   0.339227369           AI145368   0.9917   254.051109   0.364636436           AIl45614   0.9969   441.5437287   0.356577697           AI145627   0.9917   327.5608757   0.302478299           AI145853   0.9823   133.5529582   0.319436187           AI146034   0.9925   154.6275229   0.324965874           AI146037   0.9941   168.134647   0.275451237           AI146090   0.9967   305.2987201   0.284411247           AI146170   0.9944   210.5029925   0.228534495           AI168933   0.9927   219.3020558   0.278034578           AI168950   0.99    318.010511   0.362293659           AI168974   0.9754   214.4003991   0.377276222           AI168979   0.9801   212.2729809   0.331980427           AI168986   0.9746   156.7979716   0.360198438           AI169063   0.9964   290.6780252   0.385981295           AI169154   0.9968   336.5242284   0.307023873           AI169170   0.9979   769.7878541   0.398738752           AI169269   0.977    137.357114   0.35503002            AI169272   0.9696   80.83140252   0.339825829           AI169343   0.9727   166.989764   0.268576719           AI169377   0.9889   180.0298019   0.390844085           AI169461   0.9973   866.2081039   0.336846289           AI169611   0.9986   503.4638109   0.36365031            AI169615   0.9985   663.3604215   0.326765274           AI169641   0.9962   363.828376   0.277445228           AI169642   0.9856   143.4258646   0.275093398           AI170247   0.9568   102.1625518   0.31850578            AI170265   0.9961   361.1879451   0.39819102            AI170357   0.9719   133.7080641   0.281598384           AI170388   0.9935   162.5694081   0.354744306           AI170400   0.9508   75.04534008   0.377930524           AI170414   0.9824   281.1240432   0.292176861           AI170532   0.9979   325.7623378   0.256357803           AI170663   0.9919   340.0625768   0.39841173            NM_032079   0.9912   212.7965304   0.383477936           AI170780   0.9978   403.7354889   0.31491612            AI170797   0.9898   362.0104956   0.367765173           AI170807   0.9943   244.3697528   0.250841845           AI170821   0.9835   115.6515135   0.35887866            AI171212   0.9978   775.9022683   0.275974842           AI171230   0.9719   69.49621762   0.348752498           AI171232   0.9996   746.0904006   0.390223181           AI171272   0.9961   584.7944874   0.273294529           AI171273   0.9838   409.6569296   0.341742937           AI171314   0.9894   690.4176735   0.399646269           AI171345   0.9857   121.9008899   0.300431813           NM_030836   0.9942   222.0517603   0.339780512           AI171561   0.9974   913.0878863   0.23190465            NM_019208   0.9812   125.3500482   0.338940828           AI171661   0.9675   108.2601624   0.280616401           AI171737   0.9904   251.8042864   0.37508985            AI171764   0.9973   487.4162473   0.277969318           AI171768   0.9941   333.6968205   0.399552284           AI171781   0.9916   195.2329285   0.325496907           AI171783   0.9935   277.3722053   0.390225646           AI171798   0.9511   96.82212997   0.357869848           AI171809   0.9786   121.0932339   0.375796802           AI171870   0.9849   205.0661444   0.333133061           AI171882   0.9965   253.5176312   0.301825594           AI171951   0.991    200.0156482   0.247113526           AI171952   0.9979   575.4556191   0.295443738           AI171953   0.9927   553.6106997   0.357140612           AI172001   0.982    118.9182618   0.358781789           AI172069   0.9579   55.27189598   0.301195             AI172074   0.9837   135.6336179   0.35943329            AI172092   0.9622   108.3322689   0.317645185           AI172105   0.9964   431.8804655   0.3638466            AI172106   0.9559   84.1857301   0.340208075           AI172196   0.9848   219.3575094   0.331715935           AI172214   0.9946   416.072214   0.309679658           AI172218   0.9678   136.6434257   0.298583323           AI172301   0.9895   280.4677498   0.327001975           AI172358   0.9609   229.83719   0.287010264           AI172472   0.9882   178.8898637   0.356223766           AI172537   0.9762   126.3743411   0.365833038           AI175001   0.9659   61.52159827   0.398114591           AI175008   0.9927   259.7040826   0.362558835           AI175044   0.9575   219.3801203   0.389920735           AI175266   0.9973   335.3095311   0.26393186            AI175366   0.9878   219.4067753   0.316098431           AI175467   0.9974   1050.953111   0.364080843           AI175477   0.9975   658.9995781   0.339519262           AI175512   0.999    1013.050673   0.248961012           AI175547   0.9599   86.61951632   0.316920617           NM_053969   0.9975   342.207506   0.23220273            AI175991   0.9735   93.66991174   0.324717316           AI176016   0.9896   118.3407824   0.35524637            AI176121   0.9984   1070.60159   0.328798698           AI176140   0.9985   1167.568018   0.301694674           AI176304   0.9927   123.8167239   0.335661085           AI176308   0.9965   366.1948025   0.338165832           AI176309   0.9542   86.00737984   0.344481111           AI176356   0.9946   109.3821659   0.389383607           AI176401   0.9844   124.7746569   0.350696016           AI176420   0.9925   201.397161   0.350564698           AI176491   0.9919   403.6217364   0.372574341           AI176511   0.9689   113.8307692   0.39779294            AI176581   0.9974   319.3364659   0.297959615           NM_031603   0.9949   216.3561619   0.362669512           AI176680   0.9875   447.7928097   0.319707122           AI176700   0.9947   219.7853067   0.396439967           AI176724   0.9903   209.9725455   0.302455154           AI177025   0.998    610.2210784   0.281843657           AI177104   0.9826   112.3718013   0.36436637            NM_130823   0.9867   1029.21364   0.382011417           AI177275   0.9552   151.4979672   0.387740506           AI177285   0.992    464.7912768   0.382919686           NM_053323   0.9988   1040.320182   0.36489234            AI177491   0.9963   259.4459705   0.30966825            AI177513   0.9925   309.6226866   0.340314119           AI177590   0.9662   136.1993835   0.305817502           AI177593   0.9972   754.5478523   0.336073841           NM_053798   0.9932   228.7776568   0.379213177           NM_022593   0.9964   231.7472596   0.352931313           AI177765   0.9947   242.1444179   0.376897727           AI177866   0.9944   235.3041612   0.359760014           AI177871   0.9978   493.0204781   0.36528958            AI177873   0.9732   144.5782393   0.323229199           AI177875   0.9749   169.1441977   0.327244948           AI177894   0.9978   381.7493132   0.277235768           AI177902   0.978    266.8474397   0.364559195           AI177919   0.9746   156.4655233   0.307400879           AI177921   0.9989   357.8900752   0.231216519           AI178052   0.9942   210.9919805   0.3269212            AI178239   0.9946   593.0948035   0.316485994           AI178378   0.974    113.3597499   0.35910838            AI178441   0.9693   123.977961   0.3713985            AI178503   0.9805   161.8575386   0.330391311           AI178526   0.9886   237.4170053   0.351527825           AI178644   0.9698   137.6729967   0.319376532           AI178763   0.9953   470.4968798   0.293156364           AI178830   0.9803   224.383254   0.374635776           AI178955   0.9978   647.2812159   0.338554719           AI179239   0.992    158.9663152   0.393554903           AI179243   0.9584   88.44774693   0.35176644            AI179327   0.9979   769.0504848   0.342140031           AI179329   0.9616   154.42071   0.265469568           AI179335   0.999    516.3069202   0.397506405           AI179355   0.9974   440.0164012   0.302809917           AI179356   0.999    561.1786991   0.297285533           AI179380   0.9927   471.0344443   0.399527454           AI179478   0.9899   388.0292776   0.311100554           AI179587   0.9609   181.1107877   0.27873237            AI179620   0.961    115.4729915   0.386630951           AI179636   0.9952   340.9861432   0.253360334           AI179640   0.9733   101.3470166   0.317864614           AI179711   0.9917   161.2168747   0.308572322           AI179833   0.9978   601.0236764   0.205199054           AI179840   0.972    274.1603007   0.324552252           AI179865   0.9841   437.9356753   0.284891811           AI179901   0.9957   309.429126   0.297663083           AI180015   0.9994   614.8581658   0.333863576           AI180081   0.9738   389.8384712   0.311918656           AI180108   0.9864   284.6340916   0.330955649           AI180224   0.9959   277.0615562   0.26693795            AI180259   0.9973   740.007384   0.269317518           AI180283   0.9766   336.6624044   0.383017026           AI180400   0.9968   483.0335627   0.38594082            NM_133324   0.964   108.365682   0.321158744           AI180426   0.9917   193.4211032   0.399608831           AI180441   0.9793   177.7127788   0.221639307           AI227612   0.9872   131.411593   0.379405748           AI227705   0.9973   373.9045536   0.308933462           AI227743   0.99    197.5316719   0.39445111            AI227884   0.9981   1267.180243   0.223517344           AI227887   0.9987   690.3196835   0.344995772           AI227894   0.9914   150.3145056   0.266321451           AI227962   0.9693   138.7234968   0.332020291           AI228112   0.9991   577.493851   0.329771829           AI228165   0.981    245.9051905   0.288970498           AI228249   0.9917   429.499532   0.295305029           AI228383   0.9684   118.3906252   0.308243873           AI228455   0.9592   252.5491309   0.259258531           AI228582   0.9931   244.0781278   0.299501991           AI229104   0.9973   418.4519495   0.234220274           AI229251   0.9981   1138.337459   0.262304468           AI229441   0.9967   720.1847476   0.286485755           AI229487   0.9972   326.6584951   0.278494869           AI229595   0.9949   334.9399022   0.360754811           AI229702   0.9886   220.6531992   0.314985308           NM_031342   0.9864   412.8077837   0.286440425           AI230069   0.9884   252.0236987   0.315987791           AI230073   0.9973   396.2082614   0.258575264           AI230192   0.9968   592.2203167   0.261516543           AI230248   0.9949   420.0225797   0.337867921           AI230278   0.9967   324.0160367   0.337131197           AI230308   0.9803   180.5401476   0.350570361           AI230503   0.9844   135.0925865   0.332488962           AI230635   0.9949   280.9665814   0.247155413           AI230778   0.9804   107.5929071   0.343369569           AI230912   0.9954   200.5872543   0.353036114           AI231017   0.9914   198.300742   0.381906854           AI231038   0.9956   250.0682523   0.277155064           AI231050   0.9943   410.8050546   0.253822599           AI231071   0.997    393.0335939   0.198604907           AI231201   0.9983   408.0126423   0.261904403           AI231471   0.9956   346.9078164   0.371698402           AI231491   0.9912   191.2310661   0.37822703            AI231773   0.9964   604.7876854   0.27563454            AI231785   0.9978   823.1725047   0.304029365           AI231812   0.982    210.7545364   0.282818931           AI231886   0.9978   443.3205068   0.368330282           AI232030   0.9805   402.2272212   0.353433208           AI232033   0.9926   258.5749225   0.304872148           AI232060   0.9826   129.6564971   0.320998742           AI232101   0.9941   610.1122963   0.275151496           AI232112   0.973    208.4695725   0.342289609           AI232129   0.9874   161.8677131   0.257738135           AI232159   0.9844   248.331737   0.349563487           AI232163   0.9791   499.4724254   0.367022262           NM_030586   0.9845   162.7074577   0.354117606           AI232274   0.9944   229.1965947   0.309473807           AI232296   0.9575   375.5156737   0.332664579           AI232321   0.9765   95.62859761   0.357723537           AI232354   0.9963   322.4466506   0.305484765           AI232510   0.9636   204.8272079   0.384522622           AI232639   0.953    114.8533235   0.370466228           AI232731   0.9661   207.339048   0.371840978           AI232734   0.9981   379.6275284   0.307581158           AI232800   0.9504   193.9482279   0.347453565           AI232807   0.983    197.1120336   0.309983248           AI232841   0.9903   307.2566121   0.312565038           AI232887   0.9942   204.7572949   0.363005514           AI232974   0.9922   259.9191333   0.365849096           AI232979   0.9901   232.304106   0.320933431           AI233096   0.9573   188.669731   0.368765567           AI233204   0.9956   1010.090755   0.339942787           AI233222   0.9993   768.2022698   0.307770797           AI233267   0.9768   115.1184504   0.370933612           AI233308   0.9935   151.0816592   0.349681314           AI233316   0.9941   301.9356829   0.36245711            AI233350   0.9919   228.184242   0.363111901           AI233370   0.9859   189.3310194   0.376119729           AI233698   0.9969   198.6385487   0.322074038           AI233728   0.9612   143.8504827   0.392284441           AI233915   0.9968   440.8259317   0.348972124           AI234008   0.9763   144.3922001   0.323216362           AI234040   0.9959   214.889063   0.282330926           AI234149   0.9894   147.2986378   0.346303317           AI234223   0.9943   155.5855792   0.295689739           AI234237   0.9898   128.0604113   0.365310901           AI234292   0.9666   132.0303364   0.371450337           AI234336   0.9606   108.0872625   0.348978236           AI234872   0.9933   342.8342984   0.348857143           AI234933   0.9735   437.5597637   0.362073729           AI235054   0.9805   158.1214142   0.341417567           AI235219   0.9903   372.8995033   0.398319082           AI235238   0.9975   828.1063382   0.269155653           AI235271   0.9859   210.1674784   0.269465284           AI235397   0.9937   263.135593   0.33163326            AI235403   0.9927   295.6660806   0.249674383           AI235502   0.9674   227.3319345   0.366379508           AI235508   0.9741   166.283959   0.274104484           AI235510   0.9981   1041.871028   0.289468985           NM_022518   0.9893   485.7282713   0.364274933           AI235885   0.9861   143.7381502   0.330077747           AI235901   0.9788   116.0377302   0.33953459            AI235962   0.9923   170.256446   0.228940909           AI236003   0.9911   116.7852026   0.368833607           AI236307   0.9931   647.0745083   0.3535376            AI236318   0.9905   145.0224218   0.368534279           AI236520   0.9859   230.0384121   0.310186585           AI236523   0.9693   79.93762767   0.334615459           AI236529   0.9893   267.9688613   0.215091202           AI236570   0.9972   1503.592959   0.295288772           AI236681   0.9979   434.0489709   0.388139121           AI236691   0.9938   329.1311041   0.374653742           AI236704   0.9847   87.82502754   0.361266423           AI236745   0.9936   232.0804362   0.235151157           AI236763   0.9736   114.0971841   0.323485716           AI236783   0.9988   405.5882713   0.270457401           AI236800   0.9588   130.7285204   0.364611624           AI236928   0.9889   249.3895955   0.311710968           AI237199   0.9505   96.75330112   0.385809363           AI237311   0.9975   994.9034091   0.307945865           NM_053989   0.9855   152.3314711   0.348698125           AI237700   0.9899   259.4171499   0.318929992           NM_031326   0.994    181.3514518   0.358704788           AI237861   0.9915   252.7434616   0.257047104           AI237872   0.9856   177.6381287   0.287293468           AI639425   0.9834   69.09078765   0.309131529           NM_057097   0.9897   196.9213407   0.392392697           S70803   0.9906   176.564558   0.296587753           NM_022588   0.9586   73.308782   0.367974984           NM_013221   0.9839   101.6691063   0.364721399           NM_022595   0.9955   303.915792   0.34309984            NM_053799   0.9948   362.974216   0.304148568           U53859   0.9911   598.5976337   0.357330309           NM_013050   0.9878   220.0914437   0.370223521           NM_053331   0.9996   556.6565158   0.26197082            U75392   0.9967   514.1769739   0.263076873           NM_021765   0.975    119.8855262   0.279960896           NM_017276   0.9834   371.4916806   0.349680389           Y13336   0.9959   552.6661681   0.270873633