Methods and compositions for tailing and amplifying RNA
View Patent ↗The present invention provides methods and compositions that enable tagging and amplification of targeted RNA molecules. A targeted RNA molecule is any non-polyadenylated RNA molecule including, for example, miRNA, siRNA, rRNA, tRNA, synthetic RNA, or non-polyadenylated mRNA such as mRNA from bacteria. In certain aspects, the invention provides methods and compositions for the genome-wide expression analysis of bacterial genes. Significantly, the methods enable genome-wide expression analysis in circumstances where bacterial numbers were previously too low to purify adequate amounts of RNA for DNA microarray analysis or other applications. Such methods are particularly useful for the study of bacterial gene expression during host-cell infection. The invention also provides kits for tagging and amplifying targeted RNA molecules.
1. A method for amplifying a targeted RNA in a sample comprising:
(a) altering the secondary structure of the targeted RNA by denaturing the targeted RNA or contacting the targeted RNA with a single-stranded binding protein;
(b) incubating the targeted RNA in the presence of poly(A) polymerase I of E. coli and a nucleotide under conditions that allow tailing of the targeted RNA wherein said conditions include a buffer lacking Mn 2+ and, for RNA amounts in the range of 1-100 ng, the nucleotide concentration is 5-350 μM;
(c) hybridizing the targeted RNA with a promoter-oligo-dN primer complementary to the tail of the targeted RNA;
(d) extending the promoter-oligo-dN primer using a reverse transcriptase to form a first strand complementary to the targeted RNA;
(e) synthesizing a second strand complementary to the first strand; and
(f) transcribing copies of RNA initiated from the promoter-oligo-dN primer using an RNA polymerase, wherein the RNA is complementary to the second strand.
2. The method of claim 1 , wherein altering the secondary structure of the targeted RNA comprises denaturing the targeted RNA.
3. The method of claim 2 , wherein denaturing the targeted RNA is further defined as heating the targeted RNA for at least 10 minutes at a temperature of at least about 70° C.
4. The method of claim 1 , wherein altering the secondary structure of the targeted RNA comprises contacting the targeted RNA with a single-stranded binding protein a single strand binding protein.
5. The method of claim 1 , wherein the sample comprises a mixture of eukaryotic RNA and prokaryotic RNA.
6. The method of claim 1 , wherein the targeted RNA is a non-polyadenylated mRNA; an miRNA, an siRNA, or a synthetic RNA.
7. The method of claim 6 , wherein the non-polyadenylated mRNA is a bacterial mRNA.
8. The method of claim 6 , wherein the non-polyadenylated mRNA is a degraded mRNA.
9. The method of claim 1 , wherein the sample is further defined as obtained from a cell, cell culture, a body fluid, a tissue, or an organ.
10. The method of claim 9 , wherein the tissue is a fixed tissue or a frozen tissue.
11. The method of claim 1 , wherein the sample is an environmental sample.
12. The method of claim 11 , wherein the environmental sample is a soil sample, a water sample, or an air sample.
13. The method of claim 1 , further comprising depleting polyadenylated mRNA from the sample prior to tailing the targeted RNA.
14. The method of claim 1 , further comprising depleting rRNA from the sample.
15. The method of claim 1 , wherein at least 95% of the targeted RNA is tailed.
16. The method of claim 1 , wherein the tail added on to a majority of the targeted RNA molecules comprises about 20 nucleotides to about 500 nucleotides.
17. The method of claim 1 further comprising using the amplified targeted RNA for expression analysis.
18. The method of claim 4 , wherein the single strand binding protein is T4 Gene 32 protein.
19. The method of claim 1 , wherein the promoter-oligo-dN primer is a T7-oligo-dT primer or an SP6-oligo-dT primer.
20. The method of claim 1 , wherein the RNA polymerase is a T7 RNA polymerase, SP6 RNA polymerase, or T3 RNA polymerase.
21. The method of claim 1 , wherein the reverse transcriptase is an MMLV reverse transcriptase or an avian myeloblastosis virus reverse transcriptase.
22. The method of claim 1 , wherein the reverse transcriptase is mutant reverse transcriptase having reduced or absent RNase activity.
23. The method of claim 1 , wherein the reverse transcriptase is mutant reverse transcriptase engineered to have high thermal stability tip to 70° C.
24. A method for tailing a targeted RNA in a sample comprising:
(a) denaturing the targeted RNA; and
(b) incubating the targeted RNA in the presence of poly(A) polymerase I of E. coli and a nucleotide under conditions that allow tailing of the targeted RNA wherein said conditions include a buffer lacking Mn 2+ and, for RNA amounts in the range of 1-100 ng, the nucleotide concentration is 5-350 μM.