Establishment of quantitative PCR assay technique for plasma miRNA / 国际检验医学杂志

ABSTRACT

Objective To establish a specific,stable and reliable real-time fluorescence quantitative PCR for detecting plasma mi-croRNAs(miRNAs).Methods The plasma samples from 10 healthy individuals were collected,and miRNAs was extracted using mirVanaTM PARIS kit.Exogenous cel-miR-39 and cel-miR-238 and endogenous plasma miRNAs were reversely translated by spe-cific stem-loop primers and quantified by real time fluorescence quantitative PCR.Results cel-miR-39,cel-miR-238 and miR-342-3p were amplified and quantified specifically in RNA preparations isolated from plasma samples of healthy individuals.The amplifica-tion products of cel-miR-39,cel-miR-238 and miR-342-3p showed a single melting peak at 81.44,81.62 and 82.71 ℃,respectively, without primer dimer peak or non-specific peak in all 10 cases of healthy individual plasma samples.The standard deviation(SD)of intra-assay and extra-assay of miR-342-3p was 0.13-0.20,and the coefficient of variation(CV)was 0.42%-0.66%,which sug-gesting that this detection method has a good repeatability.The levels of miR-342-3p were detected in a same plasma sample,each experiment was repeated for 5 times,and normalized by cel-miR-39 and cel-miR-238.The SD and CV of ΔCt was 0.22,1.68%,re-spectively,which indicating that cel-miR-39 and cel-miR-238 could be taken as the stable exogenous reference for the plasma miR-NAs detection by real-time fluorescence quantitative PCR.Conclusion Real-time fluorescence quantitative PCR could serve as a good platform for plasma microRNA research.