Probe/target stabilization with add-in oligo

• US 20070172841A1
• Filed: 01/25/2006
• Published: 07/26/2007
• Est. Priority Date: 01/25/2006
• Status: Abandoned Application

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Wang, Hui
• Application Number:
  US11/340,338
• Publication Number:
  US 20070172841A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2545/101 with an internal standard/control

Abstract

The invention relates to analyzing a sample containing small RNAs. In exemplary embodiments, the sample is contacted with an array in the presence of an add-in oligo. In typical embodiments, probes on the array include regions that are complementary to small RNAs and regions that are complementary to the add-in oligo. The array is then interrogated to obtain information about small RNA in the sample. Arrays and kits in accordance with the invention are also described.

Claims

1. A method of analyzing a sample comprising small RNAs, the method comprising:

contacting an array with the sample in the presence of an add-in oligo, the array comprising a set of probes and an array support, each probe comprising a target complementary region bound to the array support and an add-in oligo complementary region bound to the target complementary region, the add-in oligo complementary region bound to the array support via the target complementary region; and

interrogating the array to obtain information about small RNAs in the sample.

2. The method of claim 1, wherein the add-in oligo complementary region of every probe of the probe set has the same sequence.

3. The method of claim 1, wherein the add-in oligo has a sequence that is not complementary to the sequence of any target complementary region of probes of the probe set.

4. The method of claim 1, wherein said sample comprising small RNAs is an isolated small RNA sample.

5. The method of claim 4, wherein RNAs shorter than about 300 bases constitute at least 10% of the total RNAs in the isolated RNA sample.

6. The method of claim 4, wherein RNAs shorter than about 300 bases constitute at least 60% of the total RNAs in the isolated RNA sample.

7. The method of claim 1, wherein the small RNAs comprise microRNAs.

8. The method of claim 1, wherein the probe set comprises at least 20 different probes, each of the probes directed to a different small RNA.

9. The method of claim 1, wherein the probe set comprises at least 20 different probes, each of the probes directed to a different microRNA.

10. The method of claim 1, wherein the add-in oligo complementary region is directly bound to the target complementary region.

11. The method of claim 1, wherein the add-in oligo complementary region is directly bound to a nucleotide clamp and the nucleotide clamp is directly bound to the target complementary region.

12. The method of claim 1, said contacting done under conditions sufficient to provide for binding of the small RNAs to the probes.

13. The method of claim 12, the add-in oligo present in an amount of at least IX relative to the amount of probes present on the array.

14. The method of claim 1, wherein the add-in oligo is selected from a ribonucleic acid, a deoxyribonucleic acid, a polynucleotide incorporating at least one non-natural nucleotide, or a polynucleotide comprising a DNA sequence bound to a RNA sequence.

15. The method of claim 1, wherein the add-in oligo comprises one or more Locked Nucleoside Analogues (LNA).

16. The method of claim 1, wherein at least one of the probes of the set of probes comprises one or more Locked Nucleoside Analogues (LNA).

17. The method of claim 1, wherein the add-in oligo comprises one or more moieties selected from a ribonucleotide, a deoxyribonucleotide, a modified nucleotide, a non-natural nucleotide, a Locked Nucleoside Analogues (LNA), and combinations thereof.

18. The method of claim 1, wherein the add-in oligo comprises a hairpin structure.

19. The method of claim 1, further comprising, prior to contacting the sample with the array, labeling the small RNAs with an observable label.

20. The method of claim 1, wherein the small RNAs in the sample comprising the small RNAs have an observable label.

21. An array comprising a set of probes and an array support, each probe of the set of probes comprising a target complementary region bound to the array support and an add-in oligo complementary region bound to the target complementary region, the add-in oligo complementary region bound to the array support via the target complementary region.

22. The array of claim 21, wherein each probe of the set of probes further comprises a linker moiety bound to the array support, the target complementary region bound to the array support via the linker moiety.

23. The array of claim 21, wherein each probe of the set of probes further comprises a nucleotide clamp, a hairpin structure, or both.

24. The array of claim 21, wherein the target complementary region of each probe is about 10 to about 35 nucleotides long.

25. The array of claim 21, wherein the target complementary region of each probe is directed to a small RNA independently selected from the group consisting of a short interfering RNA (siRNA), microRNA (miRNA), tiny non-coding RNA (tncRNA), and small modulatory RNA (smRNA).

26. The array of claim 21, wherein the add-in oligo complementary region is directly bound to the target complementary region.

27. The array of claim 21, wherein the add-in oligo complementary region is directly bound to a nucleotide clamp and the nucleotide clamp is directly bound to the target complementary region.

28. A kit comprising:

an array comprising a set of probes and an array support, each probe of the set of probes comprising a target complementary region bound to the array support and an add-in oligo complementary region bound to the target complementary region, the add-in oligo complementary region bound to the array support via the target complementary region; and

an add-in oligo.