TWO-PRIMER PCR FOR MICRORNA MULTIPLEX ASSAY

摘要

The present invention provides method for amplifying a specific RNA molecule in a sample, the method comprising: (a) adding a poly(ribonucleotide) sequence to RNA molecules in the sample; (b) reverse transcribing the poly-adenylated RNA molecules using a reverse primer comprising a sequence that anneals to said poly(ribonucleotide) sequence; and (c) amplifying and detecting the cDNA molecule(s) using the same reverse primer and using a forward primer specific for the RNA molecule to be detected; wherein at least one of the forward and reverse primers comprises a hairpin primer. The invention also provides kits useful for practicing this method.

主权项

1. A method for amplifying a specific RNA molecule in a sample, the method comprising:
(a) adding a poly(ribonucleotide) sequence to RNA molecules in the sample; (b) reverse transcribing the poly(ribonucleotide)-RNA molecules using a reverse primer comprising a sequence that anneals to said poly(ribonucleotide) sequence; and (c) amplifying and detecting the cDNA molecule(s) using the same reverse primer and using a forward primer specific for the RNA molecule to be detected; wherein at least one of the forward and reverse primers comprises a hairpin primer comprising:
(i) a first nucleotide sequence specific for the RNA molecule to be detected and/or a sequence that anneals to said poly(ribonucleotide) sequence;(ii) a second nucleotide sequence at the 5′ end of the first sequence;(iii) a third nucleotide sequence or spacer at the 5′ end of the second sequence;(iv) a fourth nucleotide sequence at the 5′ end of the third sequence, the fourth sequence being complementary to the second sequence, so as to form a double-stranded duplex; and(v) means for emitting a detectable signal when the strands of the duplex are separated;wherein, when the reverse primer comprises a hairpin primer, the sequence that anneals to said poly(ribonucleotide) sequence is present in said first nucleotide sequence of the hairpin primer.