Assay and process for labeling and detection of micro rna and small interfering rna sequences

• US 20060166215A1
• Filed: 07/02/2004
• Published: 07/27/2006
• Est. Priority Date: 07/02/2003
• Status: Abandoned Application

• Current Assignee: PerkinElmer LAS, Inc. (Perkinelmer Incorporated)
• Original Assignee: PerkinElmer LAS, Inc. (Perkinelmer Incorporated)
• Inventors: DiMeo, James, Joseph, Richard
• Application Number:
  US10/563,347
• Publication Number:
  US 20060166215A1
• 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 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

A process for protecting a short RNA fragment includes labeling a short RNA fragment with a detectable platinum compound forming a labeled small RNA fragment. Resulting labeled short RNA fragment is exposed to a capture oligonucleotide. The capture oligonucleotide includes at least two replicates of a nucleotide sequence complimentary to the short RNA fragment nucleotide sequence. The labeled short RNA fragment and the captured oligonucleotide sequence are brought into contact under hybridization conditions. With hybridization, the marker moiety is detected on the hybridized labeled small RNA fragment-captured oligonucleotide conjugant. A detection array for short RNA fragment includes a substrate having multiple spots, with a first spot including a first capture oligonucleotide including at least two replicates of a nucleotide sequence complimentary to a first short RNA fragment along with an additional nucleotide sequence functioning as a universal controller or spacer. Another spot on the array includes a second capture oligonucleotide having at least two replicates of a sequence complimentary to a second short RNA fragment along with an additional sequence functioning as a universal controller or spacer. Verified small RNA fragments are also obtained after mobilizing a sequence through elution and optional platinum compound removal therefrom.

Claims

1. A process for detecting a short RNA fragment comprising:

labeling the short RNA fragment having a nucleotide sequence with a detectable platinum compound having a marker moiety to form a labeled small RNA fragment;

exposing said labeled short RNA fragment to a capture oligonucleotide comprising at least two replicates of a nucleotide sequence complementary to the nucleotide sequence of said short RNA fragment;

contacting said labeled short RNA fragment and said capture oligonucleotide to hybridization conditions; and

detecting the marker moiety upon hybridization between said labeled small RNA fragment and said capture oligonucleotide.

2. The process of claim 1 wherein said small RNA fragment is present in a mixture of in vivo synthesized RNA fragments.

3. The process of claim 1 wherein said marker moiety is selected from the group consisting of:

a fluorophore, a hapten, a radioisotope, an enzyme, an enzyme substrate, a dye, a sol, a chromophore, and an antibody.

4. The process of claim 1 wherein said capture oligonucleotide is immobilized on a solid substrate.

5. The process of claim 4 wherein said solid substrate is a microarray spotted with said capture oligonucleotide and a plurality of different capture oligonucleotides that vary in nucleotide sequence relative to said capture oligonucleotide.

6. The process of claim 1 wherein said capture oligonucleotide further comprises an additional nucleotide sequence having a function selected from the group consisting of:

universal control, a spacer, and a combination thereof.

7. The process of claim 6 wherein said additional nucleotide sequence is interspersed between said at least two replicates.

8. The process of claim 6 wherein at least two additional nucleotide sequences surround the complementary RNA nucleotide sequence of interest.

9. The process of claim 1 wherein hybridization conditions include heating said labeled short RNA fragment and said capture oligonucleotide to between 30°

and 40°

Celsius.

10. The process of claim 1 wherein detection of hybridization between said labeled short RNA fragment and said capture oligonucleotide is by fluorescence.

11. The process of claim 1 wherein detection of hybridization between said labeled short RNA fragment and said capture oligonucleotide is by signal amplification.

12. The process of claim 11 wherein the signal amplification is tyramide signal amplification.

13. The process of claim 1 further comprising the step of removing nucleotide sequences over 80 nucleotides in length prior to labeling.

14. The process of claim 1 further comprising the step of purifying said labeled short RNA fragment prior to exposure of said labeled short RNA fragment to said capture oligonucleotide.

15. A detection array for short RNA fragments comprising:

a substrate;

a first spot on said substrate comprising a first capture oligonucleotide having at least two replicates of a nucleotide sequence complementary to a first short RNA fragment and having an additional nucleotide sequence having a function selected from the group consisting of;

universal control and spacer; and

a second spot on said substrate displaced from said first spot comprising a second capture oligonucleotide having at least two replicates of a nucleotide sequence complementary to a second short RNA fragment and having an additional nucleotide sequence having a function selected from the group consisting of;

universal control and spacer.

16. The array of claim 15 wherein said substrate is glass.

17. The array of claim 15 wherein said plurality of spots includes at least 10 spots.

18. The array of claim 15 wherein said first spot has a linear dimension of from 1 to 100 microns.

19. The array of claim 15 wherein the additional nucleotide sequence of said first capture oligonucleotide is interspersed between the at least two replicates.

20. A detectable small RNA fragment comprising a small RNA fragment bound to a detectable platinum compound, said small RNA fragment immobilized on a detector array according to claim 15 or 16.

21. A method of detecting a small RNA fragment which comprises binding a detectable platinum compound to said small RNA fragment and exposing the same to a detector array of claim 15.

24. A commercial package comprising a detector array according to claim 15 and a detectable platinum compound together with instructions for the use thereof as a detector for small RNA fragments.

22-23. -23. (canceled)

25-26. -26. (canceled)