Microarray analysis of RNA

• US 7,718,365 B2
• Filed: 07/09/2005
• Issued: 05/18/2010
• 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): Kapushoc; Stephen
• Assistant Examiner(s): BHAT, NARAYAN KAMESHWAR
• Application Number:
  US11/178,064
• Publication Number:
  US 20070009915A1
• Time in Patent Office: 1,774 Days
• Field of Search: 435/6, 435/91.1, 435/91.32, 536/23.1, 536/24.3
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease

Abstract

In certain embodiments, the invention provides a method of performing an array analysis, the method including contacting a sample of RNA with an analogous DNA set to provide a DNA/RNA duplex, contacting the DNA/RNA duplex with an enzyme having a DNA:RNA nuclease activity to provide a digested RNA sample, and contacting the digested RNA sample with an array under conditions sufficient to provide for specific binding to the array. The array typically is then interrogated. Kits in accordance with the invention are also described which include an analogous DNA set and an array.

Claims

1. A method of analyzing RNAs comprising:

a) contacting a sample comprising RNAs and interfering sequences that are complementary to said RNAs with an analogous DNA set to provide a DNA/RNA duplex comprising an analogous DNA of said set and a complementary interfering sequence, wherein the analogous DNA set comprises at least one sequence comprising the same sequence of bases as an RNA of said sample, except that uracils in the sequence of said RNA are substituted with thymines, and wherein said sample is isolated from a biological source;

b) contacting the DNA/RNA duplex with an enzyme having a DNA;

RNA nuclease activity to provide a digested RNA sample;

c) contacting the digested RNA sample with an array of probes that are complementary to the analogous DNAs of said analogous DNA set under conditions sufficient to provide for specific binding of said RNA to a probe of said array; and

d) interrogating the array.

2. The method of claim 1, wherein contacting the digested RNA sample with an array results in bound RNA and unbound RNA, wherein the method further comprises, prior to interrogating the array, washing the array to remove the unbound RNA.

3. The method of claim 2, wherein interrogating the array includes detecting a signal indicating the presence of the bound RNA to give raw results and processing the raw results by one or more manipulations selected from the group consisting of subtracting a background measurement, rejecting a measurement which is below a predetermined threshold, normalizing the raw results, and forming conclusions based on the raw results.

4. The method of claim 2, wherein the bound RNA comprises an observable label, and wherein interrogating the array comprises detecting a signal from the observable label and using the signal to assess binding of the bound RNA to the array.

5. The method of claim 4, wherein the observable label is selected from a chromogenic moiety, a fluorophore, a mass label, a spin label, or a radiolabel.

6. The method of claim 4, wherein the analogous DNA set lacks the observable label.

7. The method of claim 1, further comprising, prior to contacting the digested RNA sample with array, labeling the digested RNA sample with an observable label.

8. The method of claim 1, wherein the digested RNA sample comprises two populations of nucleic acids that are distinguishably labeled, and wherein interrogating the array comprises detecting distinguishable signals from the distinguishably labeled populations of nucleic acids and using the distinguishable signals to assess binding of the distinguishably labeled populations of nucleic acids.

9. The method of claim 1, wherein the array comprises a plurality of capture agents bound to an array support, wherein each of the plurality of capture agents is specific for a small RNA.

10. The method of claim 1, wherein the array comprises a plurality of capture agents bound to an array support, wherein each of the plurality of capture agents is complementary to a sequence that is proximal to an interfering sequence.

11. The method of claim 1, wherein the conditions sufficient to provide for specific binding to the array include stringent hybridization conditions.

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

13. The method of claim 12, wherein each of the plurality of oligodeoxynucleotides is at least about 8 bases long and is up to about 100 bases long.

14. 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.

15. The method of claim 1, wherein the at least one sequence includes one or more base insertions, deletions and/or substitutions relative to the RNA.

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

17. The method of claim 1, wherein the sample of RNA comprises isolated RNA having length less than about 500 bases.

18. The method of claim 1, further comprising:

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

20. The method of claim 1, wherein said at least one sequence analogous to a small RNA is a DNA sequence which is the same as an RNA sequence of the small RNA, without any base insertions, deletions, or substitutions relative to the RNA sequence, except that T'"'"'s in the DNA sequence substitute for U'"'"'s in the RNA sequence.

21. The method of claim 1, wherein said at least one sequence analogous to said RNA is substantially analogous, over an analogous region, to said RNA.

22. The method of claim 1, wherein said sample comprises a small RNA.

19. A method for analyzing a RNA comprising:

contacting a sample of RNA comprising an interfering sequence complementary to said RNA with an oligodeoxynucleotide to provide a DNA/RNA duplex, wherein the oligodeoxynucleotide is complementary to the interfering sequence and analogous to the RNA, and wherein said oligodeoxynucleotide has the same sequence of bases as said RNA, except that uracils in the sequence of said small RNA are substituted with thymines, and wherein said sample is isolated from a biological source;

contacting the DNA/RNA duplex with an enzyme having a DNA;

RNA nuclease activity to provide a digested RNA sample;

contacting the digested RNA sample with a capture agent under conditions sufficient to provide for specific binding to the capture agent to result in the RNA bound to the capture agent; and

detecting the RNA bound to the capture agent.

23. The method of claim 19, wherein said sample comprises a small RNA.

24. The method of claim 19, wherein said oligodeoxynucleotide has a DNA sequence which is the same as an RNA sequence of the RNA, without any base insertions, deletions, or substitutions relative to the RNA sequence, except that T'"'"'s in the DNA sequence substitute for U'"'"'s in the RNA sequence.