Measurement of gene expression profiles in toxicity determination
First Claim
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1. A method of determining the toxicity of a compound, the method comprising the steps of:
- administering the compound to a test organism;
extracting a population of mRNA molecules from each of one or more tissues of the test organism;
forming a separate population of cDNA molecule from each population of mRNA molecules from the one or more tissues such that each cDNA molecule of the separate populations has an oligonucleotide tag attached, the oligonucleotide tags being selected from the same minimally cross-hybridizing set;
separately sampling each separate population of cDNA molecules such that substantially all different cDNA molecules within a sample have different oligonucleotide tags attached;
sorting the cDNA molecules of each sample by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spatially discrete regions on one or more solid phase supports;
determining the nucleotide sequence of a portion of each of the sorted cDNA molecules of each separate population to form a frequency distribution of expressed genes for each of the one or more tissues; and
determining the toxicity of the compound from the frequency distribution of expressed genes in each of the one or more tissues.
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Abstract
A method is provided for assessing the toxicity of a compound in a test organism by measuring gene expression profiles of selected tissues. Gene expression profiles are measured by massively parallel signature sequencing of cDNA libraries constructed from mRNA extracted from the selected tissues. Gene expression profiles provide extensive information on the effects of administering a compound to a test organism in both acute toxicity tests and in prolonged and chronic toxicity tests.
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Citations
20 Claims
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1. A method of determining the toxicity of a compound, the method comprising the steps of:
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administering the compound to a test organism;
extracting a population of mRNA molecules from each of one or more tissues of the test organism;
forming a separate population of cDNA molecule from each population of mRNA molecules from the one or more tissues such that each cDNA molecule of the separate populations has an oligonucleotide tag attached, the oligonucleotide tags being selected from the same minimally cross-hybridizing set;
separately sampling each separate population of cDNA molecules such that substantially all different cDNA molecules within a sample have different oligonucleotide tags attached;
sorting the cDNA molecules of each sample by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spatially discrete regions on one or more solid phase supports;
determining the nucleotide sequence of a portion of each of the sorted cDNA molecules of each separate population to form a frequency distribution of expressed genes for each of the one or more tissues; and
determining the toxicity of the compound from the frequency distribution of expressed genes in each of the one or more tissues. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method of identifying genes which are differentially expressed in a selected tissue of a test animal after treatment with a compound, the method comprising the steps of:
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administering the compound to a test animal;
extracting a population of mRNA molecules from the selected tissue of the test animal;
forming a population of cDNA molecules from the population of mRNA molecules such that each cDNA molecule has an oligonucleotide tag attached, the oligonucleotide tags being selected from the same minimally cross-hybridizing set;
sampling the population of cDNA molecules such that substantially all different cDNA molecules have different oligonucleotide tags attached;
sorting the cDNA molecules by specifically hybridizing the oligonucleotide tags with their respective complements, the respective complements being attached as uniform populations of substantially identical complements in spatially discrete regions on one or more solid phase supports;
determining the nucleotide sequence of a portion of each of the sorted cDNA molecules to form a frequency distribution of expressed genes; and
identifying genes expressed in response to administering the compound by comparing the frequencing distribution of expressed genes of the selected tissue of the test animal with a frequency distribution of expressed genes of the selected tissue of a control animal. - View Dependent Claims (15, 16, 17, 18, 19, 20)
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Specification