Two-color Real-time/End-point Quantitation of MicroRNAs (miRNAs)

Abstract

The present invention is directed to methods, reagents, kits, and compositions for detecting target polynucleotide sequences, especially small target polynucleotides such as miRNAs, between two samples. A pair of linker probes can be employed in two different reactions to query a particular species of target polynucleotide. A pair of detector probes, a single forward primer specific for the target polynucleotide, and a reverse primer can be employed in an amplification reaction to query the difference in expression level of the target polynucleotide between the two samples. In some embodiments a plurality of small miRNAs are queried with a plurality of linker probes. The plurality of queried miRNAs can then be decoded in a plurality of amplification reactions.

1 . A kit comprising;
 a reaction vessel comprising a first linker probe;   a reaction vessel comprising a second linker probe;
 wherein the first linker probe and the second linker probe comprise the same 3′ target specific portion and different sample identifying portions. 
   
   
   
       2 . The kit according to  claim 1  further comprising a reaction vessel comprising a reverse transcriptase. 
   
   
       3 . The kit according to  claim 1  further comprising a reaction vessel comprising a DNA polymerase. 
   
   
       4 . The kit according to  claim 3  further comprising a primer pair, a first detector probe, and a second detector probe, wherein the primer pair, the first detector probe, and the second detector probe are present in the same reaction vessel. 
   
   
       5 . The kit according to  claim 4  wherein the primer pair comprises,
 a forward primer specific for a small target polynucleotide, and,   a universal reverse primer, wherein the universal reverse primer is encoded in the loop of the first linker probe and the second linker probe.   
   
   
       6 . A reaction mixture comprising;
 a first linker probe hybridized to a first species of a small target polynucleotide, and a second linker probe hybridized to a second species of the small target polynucleotide, wherein the first linker probe comprises a first sample identifying portion and the second linker probe comprises a second sample identifying portion.   
   
   
       7 . The reaction mixture according to  claim 6  further comprising a reverse transcriptase. 
   
   
       8 . The reaction mixture according to  claim 6  further comprising a forward primer specific for the small target polynucleotide, and a reverse primer, wherein the reverse primer is encoded in a loop of the first linker probe and a loop of the second linker probe, and wherein the sequence of the loop of the first linker probe encoding the reverse primer is the same as the sequence of the loop of the second linker probe encoding the reverse primer.