MicroRNA and Messenger RNA Detection On Arrays

• US 20100209932A1
• Filed: 02/16/2010
• Published: 08/19/2010
• Est. Priority Date: 03/10/2006
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: SPIER, EUGENE G.
• Application Number:
  US12/706,627
• Publication Number:
  US 20100209932A1
• 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 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The present teachings provide methods for reverse transcribing, and detecting, a plurality of small nucleic acids such as micro RNAs, from the same reaction mixture as a plurality of messenger RNAs. High levels of multiplexing are provided by the use of a plurality of zip-coded stem-loop reverse transcription primers, along with an oligo-dT-promoter-containing reverse transcription primer, in the same reverse transcription reaction mixture. The resulting products can be amplified in an in vitro transcription reaction, and detected on a solid support such as an array. The present teachings also provide compositions, kits, and devices for performing and detecting the reverse transcription reactions described herein.

Claims

1. A method of detecting a plurality of short target nucleic acids, and a plurality of messenger RNAs on a same solid support, wherein each short target nucleic acid is 18-25 nucleotides in length, said method comprising;

contacting the plurality of different short target nucleic acids with a plurality of target-specific stem-loop reverse transcription primers, wherein each of the plurality of stem-loop reverse transcription primers comprises a 3′

target-specific portion, a zip-code stem, and a promoter;

contacting, in the same reaction mixture as the short target nucleic acids, a plurality of messenger RNAs with an oligo-dT-promoter-containing reverse transcription primer;

extending the plurality of reverse transcription primers and the oligo-dT-promoter-containing reverse transcription primer in a reverse transcription reaction to form a collection of reverse transcription products;

amplifying the reverse transcription products in an in vitro transcription reaction comprising an enzyme corresponding to the promoter, to form a plurality of in vitro transcription products; and

,

detecting the plurality of in vitro transcription products on the same solid support.

2. The method according to claim 1 wherein the plurality of short target nucleic acids are micro RNAs.

3. The method according to claim 1 wherein the loop of the target-specific stem-loop reverse transcription primers comprises the promoter.

4. The method according to claim 1 wherein the promoter is T7.

5. The method according to claim 1 wherein the in vitro transcription reaction comprises DIG-dUTP.

6. The method according to claim 1 wherein the plurality of short target nucleic acids comprises at least 100 different short target nucleic acid species.

7. A reaction composition comprising;

(A) a plurality of target-specific stem-loop reverse transcription primers, wherein each of the plurality of target-specific stem-loop reverse transcription primers comprises a 3′

target-specific portion, a zip-code stem, a promoter; and

,

(B) an oligo-dT-promoter-containing reverse transcription primer.

8. The reaction composition according to claim 7 wherein the plurality of target-specific stem-loop reverse transcription primers comprises at least 100 target-specific stem-loop reverse transcription primer species.

9. A kit for quantitating a plurality of short target nucleic acids, and a plurality of messenger RNAs on a single solid support, wherein each short target nucleic acid is 18-25 nucleotides in length, said method comprising;

A) a plurality of target-specific stem-loop reverse transcription primers, wherein each of the plurality of target-specific stem-loop reverse transcription primers comprises a 3′

target-specific portion, a zip-code stem, and a promoter; and

,

B) an oligo-dT-promoter-containing reverse transcription primer.

10. The kit according to claim 9 further comprising a reverse transcriptase.

11. The kit according to claim 9 further comprising dNTPs.

12. The kit according to claim 9 further comprising DIG-dUTP.

13. The kit according to claim 9 further comprising a solid support, wherein the solid support comprises a plurality of immobilized probes, wherein the plurality of immobilized probes comprises;

A) a plurality of probes complementary to, or complementary to the complement of, a plurality of messenger RNAs; and

,

B) a plurality of probes complementary to, or complementary to the complement of, a plurality of short target nucleic acids and a corresponding zip-code introduced in a reverse transcription reaction.

14. The kit according to claim 13 wherein the immobilized probes comprise PNA.

15. The kit according to claim 13 wherein the immobilized probes comprise DNA.

16. A device comprising a solid support comprising a plurality of immobilized probes, wherein the plurality of immobilized probes comprises;

A) a plurality of probes complementary to, or complementary to the complement of, a plurality of messenger RNAs; and

,

B) a plurality of probes complementary to, or complementary to the complement of, a plurality of short target nucleic acids and a corresponding zip-code introduced in a reverse transcription reaction.

17. The device according to claim 16 wherein the immobilized probes comprise PNA.

18. The device according to claim 16 wherein the immobilized probes comprise DNA.

19. The device according to claim 16 wherein the plurality of probes complementary to, or complementary to the complement of, the plurality of messenger RNAs comprise at least 1000 different probes.

20. The device according to claim 16 wherein the plurality of probes complementary to, or complementary to the complement of, the plurality of short target nucleic acids and the corresponding zip-code introduced in the reverse transcription reaction comprise at least 100 different probes.