Amplification and detection of ribonucleic acids
Compositions, methods, and kits for detecting one or more species of RNA molecules are disclosed. In one embodiment, a first adaptor and a second adaptor are ligated to the RNA molecule using a polypeptide comprising double-strand specific RNA ligase activity, without an intervening purification step. The ligated product is reverse transcribed, then at least some of the ribonucleosides in the reverse transcription product are removed. Primers are added and amplified products are generated. In certain embodiments, the sequence of at least part of at least one species of amplified product is determined and at least part of the corresponding RNA molecule is determined. In some embodiments, at least some of the amplified product species are detected, directly or indirectly, allowing the presence and/or quantity of the RNA molecule of interest to be determined.
1. A composition, comprising: a single reaction mixture containing
a plurality of RNA molecules,
a plurality of a first double-stranded nucleic acid adaptor,
a plurality of a second double-stranded nucleic acid adaptor, and
a double-strand specific RNA ligase,
wherein a first strand of the first double-stranded nucleic acid adaptor of the plurality includes at least two ribonucleosides at the 3′ end, and a second strand of the first double-stranded nucleic acid adaptor of the plurality include a first single-stranded portion at the 5′ end, and
wherein a first strand of the second double-stranded nucleic acid adaptor of the plurality includes a terminal 5′ phosphate group, and a second strand of the second double-stranded nucleic acid adaptor of the plurality include a second single-stranded portion at the 3′ end, and
wherein the first single-stranded portion contains a degenerate nucleotide sequence that hybridizes to a first region of a first RNA molecule in the plurality of RNA molecules, and
wherein the second single-stranded portion contains a degenerate nucleotide sequence that hybridizes to a second region of the first RNA molecule in the plurality of RNA molecules.
2. The composition of claim 1 , wherein the RNA ligase comprises a T4 RNA ligase 2 (Rnl2).
3. The composition of claim 1 , wherein the single reaction mixture contains a single type of RNA ligase.
4. The composition of claim 1 , wherein
the second strand of the first double-stranded nucleic acid adaptors comprises at least one ribonucleotide, or
the second strand of the second double-stranded nucleic acid adaptors comprises at least one ribonucleotide, or
the second strand of the first double-stranded nucleic acid adaptors and the second strand of the second double-stranded nucleic acid adaptors comprise at least one ribonucleotide.
5. The composition of claim 1 , wherein
the plurality of the first double-stranded nucleic acid adaptor comprises a unique sample-specific bar code sequence, or
the plurality of the second double-stranded nucleic acid adaptor comprises a unique sample-specific bar code sequence, or
the plurality of the first and the second double-stranded nucleic acid adaptor comprise a different unique sample-specific bar code sequence.
6. The composition of claim 1 , wherein
the plurality of the first double-stranded nucleic acid adaptor comprises a reporter group, or
the plurality of the second double-stranded nucleic acid adaptor comprises a reporter group, or
the plurality of the first and the second double-stranded nucleic acid adaptor comprise a different reporter group.
7. The composition of claim 6 , wherein the reporter group comprises a fluorescent reporter group.
8. The composition of claim 6 , wherein the reporter group comprises biotin.
9. The composition of claim 1 , wherein
the plurality of the first double-stranded nucleic acid adaptor comprises a binding site for an amplification primer, or
the plurality of the second double-stranded nucleic acid adaptor comprises a binding site for an amplification primer, or
the plurality of the first and the second double-stranded nucleic acid adaptor comprise a binding site for a different amplification primer.
10. The composition of claim 1 , further comprising: a DNA polymerase having both a DNA-dependent DNA polymerase activity and RNA-dependent DNA polymerase activity.
11. The composition of claim 1 , further comprising:
an RNA-dependent DNA polymerase, or
an RNA-dependent DNA polymerase and a DNA-dependent DNA polymerase.
12. The composition of claim 1 , further comprising: RNaseH.
13. The composition of claim 1 , wherein the plurality of RNA comprises total RNA.
14. The composition of claim 1 , wherein the plurality of RNA comprises mRNA.
15. The composition of claim 1 , wherein the plurality of RNA comprises enriched RNA.
16. The composition of claim 15 , wherein the enriched RNA comprises polyA+RNA.
17. The composition of claim 1 , wherein the plurality of RNA comprises RNA that is depleted of an undesirable subset of RNA.
18. The composition of claim 1 , wherein the plurality of RNA comprises fragmented RNA.
19. The composition of claim 1 , wherein the plurality of RNA comprises RNA pooled from different samples.
20. The composition of claim 1 , further comprising a plurality of internal control RNA molecules having known sequences.