Patent ID: 8399221

Claim:
A method for preparing a cDNA copy of a small RNA molecule, comprising: (a) providing a small RNA from a biological sample, wherein said RNA is from 18 to 28 nucleotides in length; (b) incubating the small RNA with an enzyme capable of catalyzing the addition of nucleotides at the 3′ end of the small RNA in the presence of a single ribonucleotide triphosphate selected from the group consisting of ATP, GTP, UTP, and CTP and at a final concentration of divalent magnesium cation between 50 millimolar and 70 millimolar in a reaction to add nucleotides to the small RNA to generate a tailed small RNA; c) annealing a DNA primer to the tailed small RNA whereby the DNA template extends from the 3′ end of the tailed small RNA, thereby providing a single stranded region of DNA that may be used to direct polymerization of deoxyribonucleotide triphosphates; and (d) incubating the annealed tailed small RNA and DNA primer in the presence of reverse transcriptase; and deoxyribonucleotide triphosphates and at a final concentration of divalent magnesium cation between 50 millimolar and 70 millimolar under conditions allowing reverse transcription into cDNA and amplification of the annealed tailed small RNA to produce an amplification product.