Quantification of Micro RNA

摘要

The present invention relates to a method for quantifying a plurality of miRNA, said method comprising the successive step of providing a sample comprising a plurality of cDNA, each cDNA corresponding to a miRNA. Then, the sample contacted with a plurality of pairs of tagged primers and a pair of universal primers, each tagged primer comprising a specific moiety and a tagged moiety. Subsequently, each cDNA is amplified by performing a first PCR amplification using a pair of tagged primers, wherein annealing step is performed at a first annealing temperature T1, to provide a plurality of DNA. Furthermore, each DNA is amplified by performing a second PCR amplification using the pair of universal primers, wherein annealing step is performed at a second annealing temperature T2. Finally, the concentration each cDNA in the sample is determined to deduce the concentration of each miRNA.

主权项

1. A method for quantifying a plurality of miRNAs, said method comprising the successive step of:
a. Providing a sample comprising a plurality of cDNAs, each cDNA corresponding to a miRNA; b. Contacting the sample with a plurality of pairs of tagged primers and a pair of universal primers, each tagged primer comprising a specific moiety and a tagged moiety, one of the specific moiety being designed for hybridizing to a cDNA and each tagged moiety being designed for hybridizing to one of the universal primers; c. Amplifying each cDNA by performing a first PCR amplification using a pair of tagged primers, wherein annealing step of the first PCR amplification is performed at a first annealing temperature T1, in order to provide a plurality of DNAs comprising the tagged moieties; d. Amplifying each DNA by performing a second PCR amplification using the pair of universal primers, wherein annealing step of the second PCR amplification is performed at a second annealing temperature T2, said second annealing temperature T2 being superior to the first annealing temperature T1; e. Determining the concentration of each cDNA in the sample to deduce the concentration of each miRNA.