Patent ID: 8192941

Claim:
A method for detecting a RNA molecule in a sample, comprising: combining the sample with at least one first adaptor, at least one second adaptor, and a polypeptide comprising double-strand specific RNA ligase activity to form a ligation reaction composition in which the at least one first adaptor and the at least one second adaptor are ligated to the RNA molecule of the sample to form a ligated product in the same ligation reaction composition, wherein the at least one first adaptor comprises: a first oligonucleotide having a length of 10 to 60 nucleotides and comprising at least two ribonucleosides on the 3′-end, and a second oligonucleotide 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 comprises: a third oligonucleotide having a length of 10 to 60 nucleotides and comprising a 5′ phosphate group, and a fourth oligonucleotide 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, wherein the single-stranded portions independently have a degenerate nucleotide sequence, or a sequence that is complementary to a portion of the RNA molecule, wherein the first and third oligonucleotides have a different nucleotide sequence; wherein the RNA molecule hybridizes with the single-stranded portion of the at least one first adaptor and the single-stranded portion of the at least one second adaptor; and combining the ligated product with i) a RNA-directed DNA polymerase, ii) a DNA polymerase comprising DNA dependent DNA polymerase activity and RNA dependent DNA polymerase activity, or iii) a RNA-directed DNA polymerase and a DNA-directed DNA polymerase, reverse transcribing the ligated product to form a reverse transcribed product, digesting at least some of the ribonucleosides from the reverse transcribed product with ribonuclease H to form an amplification template, combining the amplification template with at least one forward primer, at least one reverse primer, and a DNA-directed DNA polymerase when the ligated product is combined as in i), to form an amplification reaction composition, cycling the amplification reaction composition to form at least one amplified product, and determining the sequence of at least part of the amplified product, thereby detecting the RNA molecule.