Methods for depleting RNA from nucleic acid samples
View Patent ↗The invention relates to methods of depleting RNA from a nucleic acid sample. The RNA may be any RNA, including, but not limited to, rRNA, tRNA, and mRNA. The method is useful for depleting RNA from a nucleic acid sample obtained from a fixed paraffin-embedded tissue (FPET) sample. The method may also be used to prepare cDNA, in particular, a cDNA library for further analysis or manipulation.
1. A method of depleting target RNA molecules in a mixture of nucleic acids comprising target and non-target RNA molecules comprising:
a. contacting a nucleic acid sample with a multiplicity of DNA probes complementary to a target RNA, wherein the multiplicity of DNA probes is capable of specifically hybridizing to the entire full length sequence of the target RNA, includes at least two probes complementary to sequences that overlap one another and forms a DNA-RNA hybrid; and
b. contacting the nucleic acid sample with a ribonuclease (RNase) that specifically recognizes the DNA-RNA hybrid; wherein the target RNA in the DNA-RNA hybrid is degraded.
2. The method of claim 1 , wherein the target RNA is ribosomal RNA (rRNA).
3. The method of claim 2 , wherein the rRNA is 18S rRNA, 28S rRNA, or both.
4. The method of claim 2 , wherein the rRNA is 12S rRNA, 16S rRNA, or both.
5. The method of claim 1 , further comprising the step of contacting the nucleic acid sample with a DNase after contacting the nucleic acid sample with a ribonuclease, and wherein the DNA probes are degraded.
6. The method of claim 5 , wherein the DNase is DNase I.
7. The method of claim 1 , further comprising preparing cDNA from the nucleic acid sample.
8. The method of claim 7 , further comprising sequencing the cDNA.
9. The method of claim 7 , further comprising performing gene expression analysis on the cDNA.
10. The method of claim 1 , wherein the RNase is RNaseH.
11. The method of claim 1 , wherein the target RNA comprises less than 10% of all RNA present in the nucleic acid sample after degradation with the RNase.
12. The method of claim 1 , wherein the total RNA in the nucleic acid sample is amplified prior to contacting the nucleic acid sample with the multiplicity of DNA probes.
13. The method of claim 1 , wherein the multiplicity of DNA probes are 10 to 100 nucleotides in length.
14. The method of claim 13 , wherein the multiplicity of DNA probes are 14 to 85 nucleotides in length.
15. A method of depleting a target RNA from a nucleic acid sample comprising target and non-target RNA molecules comprising:
a. contacting the nucleic acid sample with a multiplicity of DNA probes complementary to the target RNA, wherein the nucleic acid sample is obtained from a fixed paraffin-embedded tissue (FPET) sample, wherein the multiplicity of DNA probes is capable of specifically hybridizing to the entire full length sequence of the target RNA, includes at least two probes complementary to sequences that overlap one another, and forms a DNA-RNA hybrid; and
b. contacting the nucleic acid sample with a ribonuclease (RNase) that specifically recognizes a DNA-RNA hybrid; wherein the target RNA in the DNA- RNA hybrid is degraded; and
c. depleting the target RNA molecules in the sample.
16. The method of claim 15 , wherein the target RNA is ribosomal RNA (rRNA).
17. The method of claim 16 , wherein the rRNA is 18S rRNA, 28S rRNA, or both.
18. The method of claim 16 , wherein the rRNA is 12S rRNA, 16S rRNA, or both.
19. The method of claim 15 , further comprising the step of contacting the nucleic acid sample with a DNase after contacting the nucleic acid sample with a ribonuclease, and wherein the DNA probes are degraded.
20. The method of claim 19 , wherein the DNase is DNase I.
21. The method of claim 15 , wherein the FPET sample is from a tumor.
22. The method of claim 15 , wherein the RNase is RNaseH.
23. The method of claim 15 , wherein the target RNA comprises less than 10% of all RNA present in the nucleic acid sample after degradation with the RNase.