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1. (WO2005064019) AMPLIFICATION UNIVERSELLE D'ARN FRAGMENTE
Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

WHAT IS CLAIMED IS:

1. A method for preparing fragmented RNA comprising a multiplicity of RNA species for gene expression analysis comprising the steps of:
(a) polyadenylating the fragmented RNA, and
(b) converting the polyadenylated fragmented RNA obtained in step (a) to cDNA.

2. The method of claim 1 wherein the size of the RNA species within the fragmented RNA is between about 20 bases and about 2000 bases.

3. The method of claim 1 wherein the average size of the RNA species within the fragmented RNA is about 50 and about 300 bases.

4. The method of claim 1 wherein polyadenylation is performed with E. coli polyA polymerase.

5. The method of claim 1 additionally comprising the step of deblocking the 3' termini of the fragmented RNA species prior to step (a) with a deblocking agent.

6. The method of claim 5 wherein said deblocking agent is a phosphatase enzyme.

7. The method of claim 6 wherein said phosphatase is selected from the group consisting of calf alkaline phosphatase (CIP), bacterial alkaline phosphatase, shrimp alkaline phosphatase, and variants thereof.

8. The method of claim 5 wherein said deblocking agent is a polynucleotide kinase (PNK), or a variant thereof.

9. The method of claim 8 wherein said polynucleotide kinase is T4 polynucleotide kinase (T4 PNK), or a variant thereof.

10. The method of claim 5 wherein said deblocking agent is removed prior to performing the polyadenylation step (a).

11. The method of claim 5 wherein polyadenylation step (a) is performed without prior removal of the deblocking agent.

12. The method of claim 11 wherein said deblocking agent is a polynucleotide kinase (PNK).

13. The method of claim 1 wherein the polyadenylated fragmented RNA obtained in step (a) is converted to cDNA by treatment with a reverse transcriptase and oligo-dT primers.

14. The method of claim 13 wherein said reverse transcriptase is selected from the group consisting of avian myeloblastosis virus reverse transcriptase (AMV-RT), Moloney murine leukemia virus reverse transcriptase (MMLV-RT), and recombinant heterodimeric reverse transcriptases expressed in E. coli.

15. The method of claim 14 wherein said oligo-dT primers contain an RNA polymerase promoter sequence.

16. The method of claim 15 wherein said promoter is a T7 RNA polymerase promoter.

17. The method of claim 13 wherein said polyadenylated fragmented RNA obtained in step (a) is immobilized prior to conversion to cDNA.

18. The method of claim 17 wherein said immobilization is performed on beads.

19. The method of claim 18 wherein the immobilized polyadenylated fragmented RNA is enriched prior to conversion to cDNA.

20. The method of claim 19 wherein the enrichment comprises removal of rRNA sequences by hybridization to bead immobilized complementary rRNA oligonucleotides.

21. The method of claim 13 wherein the RNA is mRNA obtained from a fixed, paraffin-embedded tissue sample.

22. The method of claim 21 wherein said tissue sample is from a tumor.

23. The method of claim 22 wherein said tumor is cancer.

24. The method of claim 23 wherein said cancer is selected from the group consisting of breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, and brain cancer.

25. The method of claim 15 further comprising the step of (c) PCR amplification using one or more cDNA species present in the cDNA obtained in step (b) as a template.

26. The method of claim 25 wherein PCR amplification comprises 40 cycles, and the first five cycles are performed at a lower annealing/extension temperature .

27. The method of claim 26 wherein PCR amplification comprises 40 cycles, and the first two cycles are performed at a lower annealing/extension temperature..

28. The method of claim 25 wherein PCR amplification comprises 40 cycles, and the first two to five cycles are performed at a temperature of about 40 °C to 58 °C.

29. The method of claim 28 wherein said early cycles are performed at a temperature of about 50 °C.

30. The method of claim 15 further comprising the steps of:
(d) converting the cDNA obtained in step (b) to double-stranded DNA; and
(e) amplifying the RNA by subjecting the double-stranded DNA obtained in step (d) to in vitro transcription with an RNA polymerase to obtain amplified complementary RNA (cRNA).

31. The method of claim 30 wherein in step (d) the cDNA is converted to double-stranded DNA using RNaseH and DNA polymerase I.

32. The method of claim 30 wherein the amplified cRNA from step (e) is used directly as a template in a gene expression profiling assay by RT-PCR.

33. The method of claim 30 wherein step (e) includes labeling of the amplified cRNA with a detectable label.

34. The method of claim 33 wherein the detectable label is biotin or a fluorescent label.

35. The method of claim 30 comprising subjecting the amplified cRNA obtained in step (e) to hybridization to a microarray.

36. The method of claim 1 wherein the polyadenylated fragmented RNA obtained in step (a) is converted to cDNA by treatment with a reverse transcriptase and extended reverse primers, and the cDNA obtained is amplified by PCR using non-extended forward and a reverse PCR primers and probes, designed based on target amplicons.

37. The method of claim 36 wherein the extended reverse primers extend 10 bases further into the amplicon than the gene specific reverse PCR primer.

38. The method of claim 36 wherein the extended reverse primers extend 20 bases further into the amplicon than the gene specific reverse PCR primer.

39. The method of claim 36 wherein the extended reverse primers extend 30 bases further into the amplicon than the gene specific reverse PCR primer.

40. The method of claim 36 wherein the extended reverse primers extend into the amplicon within 1 base of the forward PCR primer.

41. A method for enhanced cDNA synthesis comprising converting RNA to cDNA by treatment with a reverse transcriptase and extended primers, and amplifying the cDNA obtained by PCR using a forward and a reverse PCR primer and a probe, designed based on a target amplicon.

42. The method of claim 41 wherein said RNA is fragmented.

43. The method of claim 42 wherein at least part of said fragmented RNA is non-polyadenylated.

44. The method of claim 43 wherein the extended reverse primers extend 10 bases further into the amplicon than the reverse PCR primer (GSP).

45. The method of claim 43 wherein the extended reverse primers extend 20 bases further into the amplicon than the reverse PCR primer (GSP).

46. The method of claim 43 wherein the extended reverse primers extend 30 bases further into the amplicon than the reverse PCR primer (GSP).

47. The method of claim 43 wherein the extended reverse primers extend to within 1 base of the forward PCR primer.

48. A method for preparing RNA comprising a multiplicity of RNA species for gene expression analysis comprising the steps of:
(a) polyadenylating said RNA; and
(b) converting the polyadenylated RNA to cDNA by reverse transcriptase and oligo dT or oligo dT-T7 primers.

49. The method of claim 48 wherein said RNA comprises RNA species with blocked 3 '-termini.

50. The method of claim 49 comprising deblocking said blocked RNA species prior to step (a).

51. The method of claim 48 further comprising the step of using the oligo dT primed cDNA directly for gene expression analysis.

52. The method of claim 51 wherein said gene expression analysis is performed by TaqMan®.

53. The method of claim 48 further comprising the step of converting the oligo dT-T7 primed cDNA to double-stranded DNA with DNA polymerase I and RNAse H, and amplifying the double-stranded DNA with T7 RNA polymerase and rNTPs.

54. The method of claim 48 further comprising the step of converting the oligo dT-T7 primed cDNA to double-stranded DNA, and amplifying the double-stranded DNA with T7 RNA polymerase and biotinylated rNTPs.

55. A method for preparing RNA comprising a multiplicity of RNA species for gene expression analysis comprising the steps of:
(a) polyadenylating said RNA; and
(b) converting the polyadenylated RNA to cDNA by reverse transcriptase and oligo dT-T7 primers containing a T7 RNA polymerase promoter, and
(c) subjecting the double-stranded DNA obtained in step (b) to in vitro transcription with a T7 RNA polymerase to obtain amplified complementary RNA (cRNA).