Amplification and detection of ribonucleic acids

摘要

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.

主权项

1. A method for detecting an RNA molecule, comprising:
(a) forming a single ligation reaction mixture containing (i) at least one RNA molecule including at least a first and a second RNA molecule, (ii) a plurality of a first double-stranded nucleic acid adaptor, (iii) a plurality of a second double-stranded nucleic acid adaptor, and (iv) an RNA ligase, wherein a first strand of the first double-stranded nucleic acid adaptors of the plurality include at least two ribonucleosides at the 3′ end, and a second strand of the first double-stranded nucleic acid adaptors 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 adaptors of the plurality include a terminal 5′ phosphate group, and a second strand of the second double-stranded nucleic acid adaptors of the plurality include a second single-stranded portion at the 3′ end, and wherein the first and the second single-stranded portions contain a nucleotide sequence that hybridizes to a portion of the at least first RNA molecule from the mixture of different RNA molecules, and wherein the first double-stranded adaptors of the plurality, or the second double-stranded adaptors of the plurality, or the first and second double-stranded adaptors comprise a unique bar code sequence; and (b) producing a plurality of different RNA ligation product by ligating the first double-stranded adaptor to a first end of the at least one RNA molecule and ligating the second double-stranded adaptor to a second end of the at least one RNA molecule.