[Comparative analysis of RT-qPCR based methodologies for microRNA detection.]

Many pathological states are accompanied by characteristic changes in the cellular profile of microRNAs - small molecules that regulate gene expression at the posttranscriptional level. This allows us to consider miRNA as a promising class of biological markers. In the work, a direct comparison of three RT-qPCR methodologies (s-Loop, u-Elong and 2-Tail) for miRNA analysis was performed. A synthetic miRNA-451 analog was used to determine the efficiency of miRNA molecule detection and analysis of the miRNA-29b, miRNA-375 and miRNA-451 profiles in OAW42 and HT29 cell lines was carried out. By the methods of 2-Tail and s-Loop, seven different miRNA were also analyzed in 13 clinical specimens. The results of the study show that in the 2-Tail and s-Loop approaches, RT-qPCR demonstrated high reproducibility in results of miRNA analysis, and a linear dependence of the mimic миРНК-451detection efficiency in the range of 107 to 103 molecules per reaction was registered. On a number of significant criteria, the two technologies turned out to be relatively equivalent, i.e. any of them can be used as a basis for the method of clinical diagnostics.