Methods for quantifying microrna precursors

Abstract

The present invention is directed to methods, reagents, kits and compositions for identifying and quantifying microRNA (miRNA) precursor expression in a biological sample. The method uses gene-specific primers and reverse transcriptase to convert the primary miRNA precursors (pri-miRNA) and pre-miRNA precursors (pre-miRNAs) to cDNA. The method also uses amplification reactions using gene specific forward and reverse primers that are targeted to the hairpin sequence of pri- and pre-microRNA precursors to detect the expression levels of both the pri- and the pre-micoRNAs. In one embodiment, the amplification reaction is a real-time PCR wherein the level of PCR amplification products produced is related to the levels of the microRNA precursors in the biological sample. In another embodiment, a probe is used to distinguish between similar isoforms of microRNA precursors. In another embodiment, the expression levels of a pre-miRNA precursor is calculated by using primers and amplification reactions that detect the pri-miRNA together with amplification reactions and primers that detect both pri- and pre-miRNAs, and calculating the difference.

1 . A method for identifying the expression of both pri-microRNA and pre-microRNA precursors in a sample, said method comprising the initial step of using a gene-specific reverse primer to reverse transcribe a target nucleotide sequence, wherein the target nucleotide sequence comprises a substantial portion of a hairpin sequence shared by both the pri- and the pre-microRNA. 
     
     
         2 - 34 . (canceled)