Compositions and Methods for the Detection of Small RNA

• US 20080131878A1
• Filed: 12/05/2006
• Published: 06/05/2008
• Est. Priority Date: 12/05/2006
• Status: Abandoned Application

• Current Assignee: Asuragen, Inc.
• Original Assignee: Asuragen, Inc.
• Inventors: Latham, Gary J., Kemppainen, Jon
• Application Number:
  US11/567,082
• Publication Number:
  US 20080131878A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA

Abstract

Embodiments of the invention include methods of detecting one or more RNA by reverse transcribing one or more RNA target using one or more reverse transcription primer comprising in a 5′ to 3′ direction (i) a primer segment, (ii) a probe segment distinct from the primer segment, and (iii) a 3′ target specific segment that anneals to a RNA target; amplifying one or more RNA from the reverse transcription reaction using a first amplification primer that anneals to the 3′ end of a reverse transcribed RNA target and a second primer that anneals to a sequence complementary to the primer segment; and detecting amplification of a target nucleic acid.

Claims

1. A method of detecting one or more RNA comprising the steps of:

(a) reverse transcribing one or more RNA target using one or more linear reverse transcription primer comprising in a 5′

to 3′

direction

(i) a primer segment,

(ii) a non-target probe segment, and

(iii) a 3′

target specific segment that anneals to a RNA target;

(b) amplifying one or more RNA or RNA segment from all or part of the reverse transcription reaction using a first amplification primer that anneals to the 3′

end of a reverse transcribed RNA target and a second primer that anneals to a sequence complementary to the primer segment; and

(c) detecting amplification of a target nucleic acid.

2. The method of claim 1, wherein the RNA target is present in less than 1,000,000 copies.

3. The method of claim 2, wherein the RNA target is present in less than 1,000 copies.

4. The method of claim 1, wherein the 3′

target specific segment of the reverse transcription primer anneals to a contiguous sequence of 7 or more nucleotides of the RNA target.

5. The method of claim 1, wherein the first amplification primer is present at a concentration of 100 to 1000 nM and the second amplification primer is present at a concentration of 100 to 1000 nM.

6. The method of claim 1, wherein detecting amplification comprises detecting association of a probe with a sequence of the probe segment or a complement to the probe segment.

7. The method of claim 6, wherein the probe is a 5′

-exonuclease assay probe, stem-loop molecular beacon, stemless or linear beacon, PNA Molecular Beacon, linear PNA beacon, non-FRET probe, stem-loop and duplex scorpion probe, bulge loop probe, pseudo knot probe, cyclicon, minor groove binding (MGB) probe, hairpin probe, peptide nucleic acid (PNA) light-up probe, self-assembled nanoparticle probe, or ferrocene-modified probe.

8. The method of claim 7, wherein the probe is a 5′

exonuclease probe or a beacon probe.

9. The method of claim 1, wherein the RNA is greater than 500 base pairs in length.

10. The method of claim 1, wherein the RNA is 1 kilobase or less in length.

11. The method of claim 10, wherein the RNA is 100 bases or less in length.

12. The method of claim 11, wherein the RNA is 25 bases or less in length.

13. The method of claim 1, wherein the first amplification primer comprises a 5′

non-complementary sequence of 2 to 10 nucleotides.

14. The method of claim 13, wherein the first amplification primer comprises a 5′

non-complimentary sequence of 4 to 8 nucleotides.

15. The method of claim 14, wherein the non-complementary sequence comprises cytosine and guanine residues.

16. The method of claim 1, further comprising diluting the reverse transcription reaction prior to amplification.

17. The method of claim 16, wherein the reverse transcription reaction is diluted 2 to 100 fold.

18. The method of claim 1, wherein the RNA is from a biopsy sample, a histological sample, or a biological fluid.

19. The method of claim 18, wherein the histological sample is a formalin or formaldehyde fixed paraffin embedded (FFPE) sample.

20. A method of detecting an miRNA in a sample comprising the steps of:

(a) obtaining a RNA sample;

(b) reverse transcribing one or more miRNA target in the RNA sample using one or more linear reverse transcription primer comprising in a 5′

to 3′

direction

(i) a primer segment,

(ii) a non-target probe segment, and

(iii) a 3′

target specific segment that anneals to a miRNA target;

(c) amplifying the product of the reverse transcription reaction using a first primer that anneals to the 3′

portion of a reverse transcribed target miRNA and a second primer that anneals to a sequence complementary to the primer segment; and

(d) detecting amplification of the probe segment.

21. The method of claim 20, wherein the sample is a biopsy sample, a histological sample, or a biological fluid.

22. The method of claim 21, wherein the histological sample is a FFPE sample.

23. A method of assessing a pathological condition comprising the steps of:

(a) reverse transcribing RNA in a RNA sample from a subject having, suspected of having, or at risk of developing a pathological condition using a reverse transcription primer specific for one or more RNA associated with one or more pathological condition using one or more linear reverse transcription primer comprising in a 5′

to 3′

direction

(i) a universal primer segment,

(ii) a non-target probe segment, and

(iii) a 3′

target specific segment that anneals to a RNA target;

(b) amplifying the product of the reverse transcription reaction using a first primer that anneals to the 5′

portion of a target RNA and a second primer that anneals to the universal primer segment of the reverse transcription primer; and

(c) detecting amplification of the probe segment of the reverse transcription primer.

24. The method of claim 23, wherein the RNA is a miRNA.

25. The method of claim 23, wherein the sample is a biopsy sample, a histological sample, or a biological fluid.

26. The method of claim 25, wherein the histological sample is a FFPE sample.

27-29. -29. (canceled)