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 small RNA library composition, comprising:
a multiplicity of amplified small RNA molecules, each comprising
at least one first adaptor sequence, a small RNA sequence, and at least one second adaptor sequence;
wherein the at least one first adaptor sequence comprises: a first oligonucleotide strand having a length of 10 to 60 nucleotides and comprising at least two ribonucleosides on the 3′-end, and a second oligonucleotide strand comprising a nucleotide sequence substantially complementary to the first oligonucleotide and further comprising a single-stranded 5′ portion of 1 to 8 nucleotides when the first oligonucleotide and the second oligonucleotide are duplexed,
wherein the at least one second adaptor sequence comprises: a third oligonucleotide strand having a length of 10 to 60 nucleotides and comprising a 5′ phosphate group, and a fourth oligonucleotide strand comprising a nucleotide sequence substantially complementary to the third oligonucleotide and further comprising a single-stranded 3′ portion of 1 to 8 nucleotides when the third oligonucleotide and the fourth oligonucleotide are duplexed that hybridizes to the small RNA molecule,
wherein the single-stranded portions of the first and second adaptors independently have a degenerate nucleotide sequence, and/or a sequence that is complementary to and that hybridizes to a portion of the small RNA molecule,
and wherein the first and third oligonucleotides have different nucleotide sequences.
2. The composition of claim 1 , wherein initial adaptor-small RNA molecules are formed by combining a plurality of RNA molecules with a plurality of first and second adaptors and a double stranded specific RNA ligase comprising a T4 RNA ligase 2 (Rnl2) in a single reaction mixture to form ligated small RNA adaptor molecules.
3. The composition of claim 2 , wherein the amplified small RNA molecules are formed by amplifying the small RNA-adaptor molecules.
4. The composition of claim 1 , wherein
the second strand of the first adaptors comprises at least one ribonucleotide, or
the fourth strand of the second adaptors comprises at least one ribonucleotide, or
the second strand of the first adaptors and the fourth strand of the second adaptors comprise at least one ribonucleotide.
5. The composition of claim 1 , wherein
the plurality of the first adaptor comprises a unique sample-specific bar code sequence, or
the plurality of the second adaptor comprises a unique sample-specific bar code sequence, or
the plurality of the first and the second adaptor comprise a different unique sample-specific bar code sequence.
6. The composition of claim 1 , wherein
the plurality of the first adaptor comprises a reporter group, or
the plurality of the second adaptor comprises a reporter group, or
the plurality of the first and the second 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 adaptor comprises a binding site for an amplification primer, or
the plurality of the second adaptor comprises a binding site for an amplification primer, or
the plurality of the first and the second adaptor comprise a binding site for a different amplification primer.
10. The composition of claim 1 , wherein the small RNA molecules comprises total RNA.
11. The composition of claim 1 , wherein the small RNA molecules comprises mRNA.
12. The composition of claim 1 , wherein the small RNA molecules comprises enriched RNA.
13. The composition of claim 1 , wherein the small RNA molecules comprises polyA+RNA.
14. The composition of claim 1 , wherein the small RNA molecules comprises RNA that is depleted of an undesirable subset of RNA.
15. The composition of claim 1 , wherein the small RNA molecules comprises fragmented RNA.
16. The composition of claim 1 , wherein the small RNA molecules comprises RNA pooled from different samples.
17. The composition of claim 1 , further comprising a plurality of internal control RNA sequence molecules having known sequences.