NOVEL METHODS FOR QUANTIFICATION OF MICRORNAS AND SMALL INTERFERING RNAS

• US 20110076675A1
• Filed: 06/01/2009
• Published: 03/31/2011
• Est. Priority Date: 04/07/2004
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Exiqon A/S (Qiagen NV)
• Inventors: Kongsbak, Lars, Kauppinen, Sakari, Echwald, Søren Morgenthaler, Nørholm, Mikkel, Nielsen, Peter Stein, Jacobsen, Nana, Mouritzen, Peter
• Granted Patent: US 8,383,344 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2561/101 Taqman

Abstract

The invention relates to ribonucleic acids, probes and methods for detection, quantification as well as monitoring the expression of mature microRNAs and small interfering RNAs (siRNAs). The invention furthermore relates to methods for monitoring the expression of other non-coding RNAs, mRNA splice variants, as well as detecting and quantifying RNA editing, allelic variants of single transcripts, mutations, deletions, or duplications of particular exons in transcripts, e.g., alterations associated with human disease such as cancer. The invention furthermore relates to methods for detection, quantification as well as monitoring the expression of deoxy nucleic acids.

Claims

1. A method for quantitative determination of a short-length RNA, which has a length of at most 100 nucleotides, comprising the steps of:

a) preparing, from a sample comprising said short-length RNA, a template polynucleotide which consists of

     1) a single stranded target sequence consisting of the sequence of said short-length RNA, its corresponding DNA sequence or a nucleotide sequence complementary to the sequence of said short-length RNA and

     2) a 5′ and

/or a 3′

adjacent nucleotide sequence by appending said 5′ and

/or a 3′

adjacent nucleotide sequence to said single stranded target sequence;

b) using said template polynucleotide in a reverse transcription or a nucleotide polymerization to obtain a strand of cDNA; and

c) performing a quantitative real-time PCR (qPCR) including as template(s) said cDNA and optionally the template polynucleotide.

2. The method of according to claim 1, wherein the 5′

and/or the 3′

adjacent nucleotide sequence is a polynucleotide consisting of identical nucleotides.

3. The method according to claim 1, wherein the 5′

and/or the 3′

adjacent nucleotide sequence(s) are covalently joined to the single stranded target sequence.

4. The method according to claim 1, wherein the single stranded target sequence and the 5′

and/or the 3′

adjacent nucleotide sequence(s) are non-covalently joined.

5. The method according to claim 1, wherein the 5′

and/or the 3′

adjacent nucleotide sequence is joined to the single stranded target sequence by an enzymatic reaction.

6. The method according to claim 1, wherein the 5′

and/or the 3′

adjacent nucleotide sequence is joined to the single stranded target sequence by a non-enzymatic reaction.

7. The method according to claim 1, wherein the 5′

and/or the 3′

adjacent nucleotide sequence does not occur naturally in the organism from where the sample RNA is derived.

8. The method according to claim 1, wherein step (a) comprises preparation of the template polynucleotide by ligation of the 5′

and/or the 3′

adjacent nucleotide sequence to the short-length RNA, or wherein step (a) comprises preparation of the template polynucleotide by joining the 5′ and

/or the 3′

adjacent nucleotide sequence to the short-length RNA in a terminal transferase reaction, wherein the ligation is selected from overhang ligation and blunt-end ligation.

9. The method according to claim 8, wherein the ligation is overhang ligation.

10. The method according to claim 8, comprising annealing, to the short-length RNA, an oligonucleotide in part complementary to the ligase-reactive end of the 5′

or the 3′

adjacent nucleotide sequence and in part complementary to the ligase reactive end of the short-length RNA molecule so as to position the ligase-reactive end of the 5′

or the 3′

adjacent nucleotide sequence directly adjacent to the ligase-reactive end of the small RNA molecule to allow overhang ligation.

11. The method according to claim 8, wherein all RNA in the sample is subjected to the ligation or terminal transferase reaction.

12. The method according to claim 8, wherein ligation to the 5′

end of the target sequence is performed by phosphorylating the 5′

end of the target sequence prior to the ligation reaction.

13. The method according to claim 8, wherein the 5′

adjacent nucleotide sequence is blocked at its 5′

terminus and the 3′

adjacent nucleotide sequence is blocked at its 3′

terminus prior to ligation.

14. The method according to claim 1, wherein the 5′

and/or the 3′

adjacent nucleotide sequence is preferentially or exclusively joined to a defined processing state of said short-length RNA in step (a).

15. The method according to claim 14, wherein the defined processing state of said RNA is the mature state.

16. The method according to claim 8, wherein step (b) comprises reverse transcription of the template polynucleotide to obtain the cDNA.

17. The method of claim 8, wherein said terminal transferase reaction is a poly-A transferase reaction.

18. The method according to claim 1, wherein step (a) comprises a step of nucleotide polymerization to attach the adjacent nucleotide sequences.

19. The method according to claim 18, wherein the polymerization is achieved by means of a polymerase selected from the group consisting of a template-independent and a template-dependent polymerase.

20. The method according to claim 19, wherein the polymerase is a DNA polymerase.

21. The method according to claim 1, wherein the polymerization consists in addition of a poly-A, poly-G, poly-T or a poly-C tail to the 3′

end of the target sequence.

22. The method according to claim 18, wherein step (a) comprises preparation of the template polynucleotide by the steps of:

annealing the 3′

end of the short-length RNA to an oligonucleotide capture probe the 5′

end of which is complementary to the 3′

end of the short-length RNA; and

extending the short-length RNA by nucleotide polymerization using the oligonucleotide capture probe as template so as to obtain an extended short-length RNA molecule which constitutes the template polynucleotide.

23. The method according to claim 22, where the nucleotide polymerisation comprises a DNA polymerisation to so as to obtain an RNA-DNA hybrid which constitutes the template polynucleotide.

24. The method according to claim 22, wherein step (b) comprises that the RNA-DNA hybrid strand is reverse transcribed to obtain the cDNA, optionally after removal of material not annealing to the oligonucleotide capture probes.

25. The method according to claim 24, wherein the primer in the reverse transcription is the oligonucleotide capture probe or a separate reverse transcription primer.

26. The method according to claim 18, wherein step (a) comprises preparation of the template polynucleotide by the steps of:

annealing the 5′

end of the short-length RNA to an oligonucleotide capture probe the 3′

end of which is complementary to the 5′

of the short-length RNA and the 5′

end of which comprises the 5′

adjacent nucleotide sequence; and

extending the capture probe by reverse transcription using the short-length RNA as template to obtain an extended capture probe constituting the template polynucleotide.

27. The method according to claim 26, wherein step (b) comprises that the short-length RNA is removed from the extended capture probe, the capture probe is allowed to anneal at its 3′

end to a helper oligonucleotide comprising a nucleotide sequence complementary to the 3′

adjacent nucleotide sequence, and the capture probe is further elongated in the 5′

→

3′

direction to obtain the cDNA by means of DNA polymerization using the helper oligonucleotide as template.

28. The method according to claim 26, wherein the capture oligonucleotide contains a moiety that enables immobilisation onto a solid support.

29. The method according to claim 28, wherein the capture probe is immobilised after annealing so as to allow removal of non-annealing material.

30. The method according to claim 1, wherein the sample in step (a) is enriched for RNA of short length.

31. The method according to claim 1, wherein step (c) comprises use of a detection probe which comprises modified nucleotides.

32. The method according to claim 31, wherein the modified nucleotides are LNA nucleotides.

33. The method according to claim 31, wherein the detection probe corresponds to or is complementary to a sequence in the short-length RNA.

34. The method according to claim 1, wherein primers used in reverse transcription or in DNA polymerization comprise modified nucleotides.

35. The method according to claim 34, wherein the modified nucleotides are LNA nucleotides.

36. The method according to claim 1, wherein at least one primer used in the qPCR in step (c) is constituted by a primer used in the reverse transcription or nucleotide polymerization of step (b).