[Isolation of extracellular micro-vesicles from cell culture medium: comparative evaluation of methods]. / Sravnitel'nyi analiz metodov vydeleniia vnekletochnykh mikrovezikul iz kul'tural'noi sredy.

Extracellular vesicles (EV) are secreted by cells of multicellular organisms. EV mediate specific mode of intercellular communication by "horizontal" exchange of substances and information. This phenomenon seems to have an essential biological significance and became a subject of intensive research. Biogenesis, structural and functional features of the EV is being commonly studies in in vitro condition. Several methods of EV isolation from cell culture medium are established, however selection of method might influence on obtained results. The choice of the optimal method depends usually from the amount of medium and the aims of the research while is still challenging issue. We performed a comparative analysis of four different methods of EV isolation from cell culture medium: differential ultracentrifugation, ultracentrifugation with a 30% sucrose/D2O "cushion", precipitation with plant proteins and immune-affinity capturing. EV isolated by different approaches were compared in terms of following parameters: size, concentration, morphology of EV, contamination by non-vesicular particles, content of exosomal tetraspanins on the EV surface, content of total proteins, RNA, and several glioma-associated miRNAs. Applied methods included nano-patricle tracking analysis (NTA), dynamic light scattering (DLS), cryo-electron microscopy, flow cytometry and RT-qPCR. On the base of obtained results, we developed practical recommendations that may help researchers to make a best choice of EV isolation method.

[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.