Method of treatment of RNA sample

• US 8,076,064 B2
• Filed: 07/09/2005
• Issued: 12/13/2011
• Est. Priority Date: 07/09/2005
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Wang, Hui
• Primary Examiner(s): Lu, Frank W
• Application Number:
  US11/177,679
• Publication Number:
  US 20070009913A1
• Time in Patent Office: 2,348 Days
• Field of Search: 435/6, 435/91.1, 435/91.21, 435/91.51, 435/183, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.1, 536/25.2, 536/25.3
• US Class Current: 435/6
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2320/10 in screening processes

Abstract

The invention relates to methods for treating samples of RNA. In an embodiment the method includes contacting the sample of RNA with an analogous DNA set to provide a DNA/RNA duplex. The analogous DNA set includes at least one sequence analogous to a small RNA. The method includes contacting the DNA/RNA duplex with an enzyme having a DNA:RNA nuclease activity to provide a digested RNA sample. Kits in accordance with the invention are also described.

Claims

1. A method of treating a sample of RNA, the method comprising:

a) contacting the sample of RNA with an analogous DNA set to provide a DNA/RNA duplex, wherein the analogous DNA set comprises at least one DNA oligonucleotide comprising at least ten contiguous nucleotides of a small RNA in said sample, except that uracils in the sequence of said small RNA are substituted with thymines in said DNA oligonucleotide;

b) contacting the DNA/RNA duplex with an enzyme having an RNase H activity to provide a digested RNA sample; and

c) analyzing said digested RNA sample to detect said small RNA.

2. The method of claim 1, further comprising:

contacting the digested RNA sample with an enzyme having a DNA nuclease activity to result in digestion of the analogous DNA set.

3. The method of claim 2, wherein said enzyme having a DNA nuclease activity is DNase I.

4. The method of claim 1, wherein said small RNA is selected from the group consisting of a short interfering RNA (sRNA), microRNA (miRNA), tiny non-coding RNA (tncRNA) and a small modulatory RNA (smRNA).

5. The method of claim 1, further comprising inactivating the enzyme having the RNase H activity after the digested RNA sample is provided.

6. The method of claim 1, further comprising separating the enzyme having the RNase H activity from the digested RNA sample.

7. The method of claim 1, wherein the analogous DNA set comprises a plurality of different oligodeoxynucleotides, each of the plurality of different oligodeoxynucleotides comprising a sequence analogous to a small RNA.

8. The method of claim 7, wherein each of the plurality of different oligodeoxynucleotides is at least about 8 bases long to about 100 bases long.

9. The method of claim 7, wherein each of the plurality of different oligodeoxynucleotides is at least about 10 bases long to about 50 bases long.

10. The method of claim 1, wherein the analogous DNA set comprises at least 5 different oligodeoxynucleotides, each of the oligodeoxynucleotides comprising a sequence analogous to a small RNA.

11. The method of claim 1, wherein the analogous DNA set comprises at least one oligodeoxynucleotide comprising a sequence analogous to a small RNA, wherein the sequence includes one or more base insertions, deletions and/or substitutions relative to the small RNA.

12. The method of claim 1, wherein the analogous DNA set comprises at least one polydeoxynucleotide having a plurality of small RNA complementary sequences.

13. The method of claim 1, wherein the enzyme having the RNAse H activity is RNase H.

14. The method of claim 1, wherein the enzyme having the RNAse H activity is a thermostable RNase H.

15. The method of claim 1, further comprising, prior to contacting the sample of RNA with the analogous DNA set, labeling the sample of RNA with an observable label.

16. The method of claim 1, further comprising labeling the digested RNA sample with an observable label.

17. The method of claim 1, wherein the sample of RNA comprises RNA that has an observable label attached thereto.

18. The method of claim 14, wherein the analogous DNA set lacks the observable label.