Materials and methods for profiling micrornas

Abstract

The present invention provides materials and methods for detecting, quantifying, and/or high-throughput-profiling microRNAs. Advantageously, the present invention is more sensitive and specific than other currently-available miRNA qPCR assays. In addition, the present invention is convenient, easy-to-perform, and cost-effective. In one embodiment, the present invention provides a universal primer for reverse transcription of all miRNAs, a universal reverse primer for PCR amplification reaction, and universal probes. In another embodiment, the present invention provides assays that allow simultaneous detection and/or quantification of a plurality of target miRNAs using a single reverse transcription reaction.

We claim: 
     
         1 . A method for detecting, quantifying, and/or profiling a target miRNA, comprising:
 a) contacting a sample containing miRNAs with an effective amount of poly(A)polymerase molecules to yield 3′ end-polyadenylated miRNA molecules,   b) contacting the sample with an effective amount of a universal primer for reverse transcription and reverse transcriptases, and reverse transcribing the polyadenylated miRNA molecules to yield corresponding c-DNA molecules; and   c) contacting the sample with an effective amount of a universal reverse primer and a target miRNA-specific forward primer, and amplifying the corresponding c-DNA molecules using an amplification reaction;   wherein the universal primer for reverse transcription is an oligonucleotide comprising: a (dT) n  sequence flanked by a stem-looped universal adaptor sequence, wherein “n” is an integer ranging from 8 to 50, wherein the universal primer comprises at least two nucleotides adjacent to the 3′ end of the (dT) n  sequence, and the nucleotide immediately adjacent to the (dT) n  sequence is not T, wherein the universal adaptor sequence near the 5′ end of the (dT) n  sequence forms into a stem-loop structure by base-pairing, and   wherein the universal reverse primer in the amplification reaction is an oligonucleotide comprising a sequence that is, or base-pairs with, at least part of the adaptor sequence near or toward the 5′ end of the (dT) n  sequence.   
     
     
         2 . The method, according to  claim 1 , wherein the forward primer comprises the target miRNA sequence and three or more additional nucleotides attached to the 3′ end of the target mature miRNA sequence. 
     
     
         3 . The method, according to  claim 1 , wherein the additional nucleotides attached to the 3′ end of the target mature miRNA sequence are adenine molecules. 
     
     
         4 . The method, according to  claim 2 , wherein one or more additional nucleotides are attached to the 5′ end of the target mature miRNA sequence. 
     
     
         5 . The method, according to  claim 1 , wherein the amplification reaction for cDNA amplification is quantitative real time polymerase chain reaction (qRT-PCR). 
     
     
         6 . The method, according to  claim 1 , comprising:
 contacting the sample with a sufficient amount of a universal probe,   detecting and/or quantifying a level of amplified c-DNA molecules, and   determining the level of the target miRNA based on the c-DNA level;   wherein the universal probe comprises a sequence that is, or base-pairs with, at least part of the adaptor sequence near the 5′ end of the (dT) n  sequence.   
     
     
         7 . The method, according to  claim 6 , wherein the universal probe comprises locked nucleic acid (LNA) molecules. 
     
     
         8 . The method, according to  claim 6 , wherein the universal probe comprises a detectable moiety. 
     
     
         9 . The method, according to  claim 8 , wherein the detectable moiety is a fluorophore selected from FAM, CY5, CY3, BODIPY FL, TEXAS RED, or any combination of two or more of the foregoing. 
     
     
         10 . The method, according to  claim 6 , wherein at least two universal probes are used. 
     
     
         11 . The method, according to  claim 1 , wherein the amplification reaction for amplification of cDNA molecules is performed once. 
     
     
         12 . The method, according to  claim 1 , wherein the threshold cycle (Ct) of the amplification reaction for amplification cDNA molecules ranges from 17 to 35. 
     
     
         13 . The method, according to  claim 1 , wherein the sample is a total RNA sample. 
     
     
         14 . The method, according to  claim 1 , wherein the method is capable of detecting, quantifying and/or profiling microRNA in a total RNA sample of about 1 pg. 
     
     
         15 . The method, according to  claim 1 , wherein the universal reverse primer comprises dUTP. 
     
     
         16 . The method, according to  claim 15 , further comprising adding uracil-DNA Glycosylase molecules to the amplification reaction mixture. 
     
     
         17 . The method, according to  claim 1 , comprising adding nuclease molecules to the reaction mixture.