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 - 27 . (canceled) 
     
     
         28 . A kit comprising at least one gene-specific reverse primer targeted to a first sequence within a hairpin sequence, wherein said hairpin sequence is shared by both a pri-microRNA precursor and its corresponding pre-microRNA precursor and the at least one gene-specific reverse primer binds substantially within the hairpin sequence. 
     
     
         29 . The kit of  claim 28 , further comprising at least one forward primer targeted to a second sequence within the hairpin sequence, wherein the forward and the reverse primer are capable of amplifying a target nucleotide sequence that comprises a portion of the hairpin sequence and both primers bind substantially within the hairpin sequence. 
     
     
         30 . The kit of  claim 28 , wherein the forward primer is targeted to the 5′ end of the hairpin sequence and the reverse primer is targeted to the 3′ end of the hairpin structure 
     
     
         31 . The kit of  claim 28 , further comprising at least one oligonucleotide probe targeted to a third sequence within the hairpin sequence, wherein said third sequence lies in between the first and the second sequences. 
     
     
         32 . The kit of  claim 31 , wherein the probe is a molecular beacon probe or an exonuclease probe. 
     
     
         33 . The kit of  claim 32 , wherein the exonuclease probe is a TaqMan probe. 
     
     
         34 . A kit for detecting a pre-microRNA precursor in a sample containing the pre-microRNA precursor and its corresponding pri-microRNA precursor, wherein said pri-microRNA and pre-microRNA precursors comprise a common hairpin sequence, the kit comprising:
 (a) a first primer targeted to a sequence that is partially upstream or downstream of the hairpin sequence; and   (b) a second primer targeted to a sequence within the hairpin sequence,   
       wherein both primers bind substantially within the hairpin sequence.