Method for identification and quantification of short or small RNA molecules

• US 20060063181A1
• Filed: 08/15/2005
• Published: 03/23/2006
• Est. Priority Date: 08/13/2004
• Status: Abandoned Application

• Current Assignee: Solexa Incorporated (Illumina Incorporated), University of Delaware
• Original Assignee: Solexa Incorporated (Illumina Incorporated), University of Delaware
• Inventors: Lu, Cheng, Luo, Shujun, Mayers, Blake, Green, Pamela J., Haudenschild, Christian D.
• Application Number:
  US11/204,903
• Publication Number:
  US 20060063181A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads

Abstract

A method of identifying and quantifying small RNA molecules comprising a) isolating RNA molecules; b) ligating RNA adapter molecules onto the isolated RNA molecules to form RNA template molecules; c) forming complementary DNA molecules by transcribing the RNA template molecules; d) amplifying the complementary DNA molecules; e) obtaining sequence information of the complementary DNA molecules (and thereby the RNA from which it was derived); and f) obtaining quantity information of the complementary DNA molecules, wherein the quantity information of the DNA molecules reflects the quantity of the isolated RNA molecules is provided. Included in the invention is the identification of RNA molecules between 15 and 30 nucleotides in length.

Claims

1. A method of identifying and quantifying RNA molecules within a population of isolated RNA molecules, the method comprising:

a) ligating RNA adapter molecules onto the isolated RNA molecules to form RNA template molecules;

b) forming complementary DNA molecules by transcribing the RNA template molecules;

c) amplifying the complementary DNA molecules;

d) obtaining sequence information of the complementary DNA molecules; and

e) obtaining quantity information of the complementary DNA molecules, wherein the quantity information of the complementary DNA molecules reflects the quantity of the isolated RNA molecules.

2. The method of claim 1 wherein the isolated RNA molecules are isolated by gel electrophoresis.

3. The method of claim 1 wherein the isolated RNA molecules are isolated by size.

4. The method of claim 1 wherein the isolated RNA molecules are about 600 nucleotides or less in length.

5. The method of claim 1 wherein the isolated RNA molecules are between about 21 and about 24 nucleotides in length.

6. The method of claim 1 wherein the step of ligating RNA adapter molecules onto the isolated RNA molecules comprises ligating a 5′

adapter sequence and a 3′

adapter sequence onto the isolated RNA molecules.

7. The method of claim 6 wherein the method comprises purifying the RNA template molecules after ligating the 5′

adapter sequence onto the isolated RNA molecules.

8. The method of claim 6 wherein the method comprises purifying the RNA template molecules after ligating the 3′

adapter sequence onto the isolated RNA molecules.

9. The method of claim 1 wherein the RNA adapter molecules comprise a restriction enzyme recognition site and an amplification priming site.

10. The method of claim 9 wherein the RNA adapter molecules further comprise a restriction enzyme recognition site, a PCR primer recognition site, and a sequencing initiation site.

11. The method of claim 1 wherein the RNA adapter molecules further comprise an amplification priming site, functionality for covalent attachment at the terminus, and a sequencing initiation site.

12. The method of claim 1 wherein the RNA adapter molecules comprise a polynucleotide sequence of SEQ ID NO:

1.

13. The method of claim 1 wherein the RNA adapter molecules comprise a polynucleotide sequence of SEQ ID NO:

2.

14. The method of claim 1 further comprising a step of digesting the amplified complementary DNA molecules with a restriction enzyme.

15. The method of claim 14 wherein the restriction enzyme comprises SFaN1.

16. The method of claim 1 wherein the steps of obtaining sequence information and quantity information comprise performing a massively parallel signature sequencing (MPSS) method.

17. A method of identifying small RNA molecules within a population of isolated RNA molecules, the method comprising:

a) ligating RNA adapter molecules onto the isolated RNA molecules to form RNA template molecules;

b) forming complementary DNA molecules by transcribing the RNA template molecules;

c) amplifying the complementary DNA molecules; and

d) obtaining sequence information of the complementary DNA molecules.

18. A method of identifying and quantifying small RNA sequences, the method comprising:

a) isolating RNA molecules;

b) sequencing the isolated RNA molecules; and

c) identifying small RNA sequences from the sequencing data of the isolated RNA molecules

d) determining the quantity of each small RNA sequence.

19. The method of claim 18 wherein, prior to step b), further comprising the steps of:

a) ligating RNA adapter molecules onto the isolated RNA molecules to form RNA template molecules; and

b) forming complementary DNA molecules by transcribing the RNA template molecules.

20. The method of claim 19 further comprising the step of amplifying the complementary DNA molecules.