Claims
- 1. A method of analyzing an RNA sample comprising:
contacting an RNA sample with random primers under hybridization conditions; generating cDNA from the RNA sample by extending the random primers with reverse transcriptase to produce cDNA; degrading the RNA population; fragmenting the cDNA; labeling the cDNA fragments; contacting the labeled cDNA fragments with a solid support comprising nucleic acid probes under hybridization conditions; and detecting the presence or absence of hybridization of the labeled cDNA fragments to the nucleic acid probes on the solid support.
- 2. The method of claim 1 wherein, for the majority of RNAs in the starting sample, the number of cDNA copies of a given sequence near the 3′ end of a single species of RNA is not more than twice the number of cDNA copies of a given sequence near the 5′ end of said single species of RNA.
- 3. The method of claim 1 wherein said RNA is selected from the group consisting of total RNA, mRNA and poly(A)+RNA.
- 4. The method of claim 1 wherein hybridization is detected by detecting a signal from labeled DNA which is hybridized to the solid support.
- 5. The method of claim 1 wherein the cDNA fragments are labeled by the addition of at least one labeled nucleotide using terminal transferase.
- 6. The method of claim 4 wherein the signal is amplified.
- 7. The method of claim 4 wherein the amount of signal detected with a probe to a 3′ region of an RNA from the starting material is not more than twice the amount of signal detected with a probe to a 5′ region of said RNA from the starting material.
- 8. The method of claim 6 wherein the amount of signal detected with a probe to a 3′ region of an RNA from the starting material is not more than twice the amount of signal detected with a probe to a 5′ region of said RNA from the starting material.
- 9. The method of claim 1 wherein the molar amount of cDNA fragments that hybridize to a probe to a 3′ region of a RNA and the molar amount of cDNA fragments that hybridize to a probe to a 5′ region of said RNA vary by 2 fold or less.
- 10. The method of claim 1 wherein the solid support comprising nucleic acid probes is selected from the group consisting of a nucleic acid probe array, a membrane blot, a microwell, a bead, and a sample tube.
- 11. The method of claim 1 wherein the random primers are 6 nucleotides in length.
- 12. The method of claim 1 wherein the random primers are 9 nucleotides in length.
- 13. The method of claim 1 wherein the random primers are 15 nucleotides in length.
- 14. The method of claim 1 wherein the RNA sample is isolated from a prokaryotic cell.
- 15. The method of claim 1 wherein the RNA sample is isolated from a eukaryotic cell or tissue.
- 16. The method of claim 15 wherein the eukaryotic cell or tissue is mammalian.
- 17. The method of claim 16 wherein the eukaryotic cell or tissue is human.
- 18. The method of claim 1 wherein the RNA sample is isolated from a source selected from the group consisting of dissected tissue, microdissected tissue, a tissue subregion, a tissue biopsy sample, a cell sorted population, a cell culture, and a single cell.
- 19. The method of claim 1 wherein the RNA sample is isolated from a cell or tissue source selected from the group consisting of brain, liver, heart, kidney, lung, retina, bone, lymph node, endocrine gland, reproductive organ, blood, nerve, vascular tissue, and olfactory epithelium.
- 20. The method of claim 1 wherein the RNA sample is isolated from a cell or tissue source selected from the group consisting of embryonic and tumorigenic.
- 21. The method of claim 1 further comprising amplifying the cDNA fragments to produce amplified cDNA fragments.
- 22. The method of claim 21 further comprising:
contacting said amplified cDNA fragments with a solid support comprising nucleic acid probes.
- 23. The method of claim 22 further comprising:
detecting the presence or absence of hybridization of said amplified cDNA fragments to the nucleic acid probes on the solid support.
- 24. The method of claim 23 wherein the solid support is selected from the group consisting of a nucleic acid probe array, a membrane blot, a microwell, a bead, and a sample tube.
- 25. The method of claim 1 wherein the RNA sample is further contacted with primer comprising poly dT.
- 26. The method of claim 23 wherein hybridization is detected by detecting a signal from labeled DNA which is hybridized to the solid support.
- 27. The method of claim 26 wherein the signal is amplified.
- 28. A gene expression monitoring system comprising the labeled cDNA fragments of claim 1 and a solid support comprising nucleic acid probes.
- 29. A gene expression monitoring system comprising the labeled cDNA fragments of claim 21 and a solid support comprising nucleic acid probes.
- 30. A kit for the detection of nucleic acids, wherein the kit comprises a container, instructions for use, random primers, buffers, a reverse transcriptase, DNase, a terminal transferase and an a solid support comprising nucleic acid probes.
- 31. A method of detecting one or more isoforms of RNA in an RNA sample comprising:
contacting an RNA sample with random primers under hybridization conditions; generating cDNA from the RNA sample by extending the random primers with reverse transcriptase to produce cDNA; degrading the RNA population; fragmenting the cDNA; labeling the cDNA fragments; contacting the labeled cDNA fragments with an array comprising a probe that hybridizes to the complement of a sequence present in a first isoform but absent from a second isoform; and detecting the presence or absence of hybridization of the labeled cDNA fragments to the array.
- 32. The method of claim 31 wherein the RNA sample is selected from the group consisting of total RNA, poly(A)+ RNA and MRNA.
- 33. The method of claim 31 wherein the RNA sample is isolated from a eukaryotic cell or tissue.
- 34. A method of detecting one or more isoforms of RNA in an RNA sample comprising:
contacting an RNA sample with random primers under hybridization conditions; generating cDNA from the RNA sample by extending the random primers with reverse transcriptase to produce CDNA; degrading the RNA population; fragmenting the cDNA; labeling the cDNA fragments; contacting the labeled cDNA fragments with an array comprising a probe that hybridizes to the complement of a sequence common to each of the one or more isoforms; and detecting the presence or absence of hybridization of the labeled cDNA fragments to the array.
- 35. The method of claim 34 wherein the RNA sample is selected from the group consisting of total RNA, poly(A)+ RNA and MRNA.
- 36. The method of claim 34 wherein the RNA sample is isolated from a eukaryotic cell or tissue.
- 37. A method of detecting all RNA transcripts of a single gene present in an RNA sample and distinguishing between different transcript isoforms present in said RNA sample comprising:
contacting an RNA sample with random primers under hybridization conditions; generating cDNA from the RNA sample by extending the random primers with reverse transcriptase to produce cDNA; degrading the RNA population; fragmenting the cDNA; labeling the cDNA fragments; contacting the labeled cDNA fragments with an array comprising a probe that hybridizes to the complement of a sequence present in each of the one or more isoforms and a probe that hybridizes to the complement of a sequence common to each of the one or more isoforms; and detecting the presence or absence of hybridization of the labeled cDNA fragments to the array.
- 38. The method of claim 37 wherein the RNA sample is selected from the group consisting of total RNA, poly(A)+ RNA and mRNA.
- 39. The method of claim 37 wherein the RNA sample is isolated from a eukaryotic cell or tissue.
- 40. A method of detecting the presence or absence of transcriptional activity from a region of a genome comprising:
obtaining a sample of RNA transcribed from said genome; contacting said RNA sample with random primers under hybridization conditions; generating cDNA from the RNA sample by extending the random primers with reverse transcriptase; degrading the RNA; fragmenting the cDNA; labeling the cDNA fragments; contacting the labeled cDNA fragments with an array comprising probes that hybridize to a plurality of sequences present in said genome; and detecting the presence or absence of hybridization of the labeled cDNA fragments to the array.
RELATED APPLICATIONS
[0001] The present application claims priority to U.S. application Ser. No. 09/641,081 filed Aug. 16, 2000 the disclosure of which is incorporated herein by reference in its entirety for all purposes.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60205432 |
May 2000 |
US |
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
09641081 |
Aug 2000 |
US |
Child |
10090320 |
Mar 2002 |
US |