METHODOLOGY TO STUDY SINGLE EXTRACELLULAR VESICLES OF VARIOUS CELLULAR ORIGIN

Extracellular vesicles are a heterogeneous group of particles surrounded by a phospholipid bilayer and having a diameter from 30 nm to 5 microns. In recent years, the involvement of extracellular vesicles in the pathogenesis of many diseases, as well as the possibility of their use in diagnosis and therapy, has been actively studied. It is known that vesicles can carry nucleic acids, in particular micro-RNA, mRNA, etc., what often leads to their participation in the regulation of many pathological and physiological processes in the body, as well as in intercellular cooperation. The repertoire of micro-RNAs carried by vesicles can vary significantly depending on their cellular origin and the functional state of the cells. To study the role of micro-RNAs carried by extracellular vesicles, it is necessary to develop and validate new approaches for the targeted production of these objects, followed by the study of the cargo carried by them. The purpose of this study was to determine the possibilities of using the method of highly sensitive fluorescence-activated sorting to obtain single extracellular vesicles with a given phenotype from blood plasma samples. A standard number of platelet and erythrocyte vesicles with the phenotype CD41+CD235a- and CD41-CD235a+, respectively, with a ranking from 1 to 15,625 pieces per sample were obtained from blood plasma samples of healthy donors by highly sensitive fluorescence-activated sorting. The level of miR-451a, miR-199a-3p, miR-21-5p in all samples of sorted vesicles was assessed by quantitative PCR. The obtained results indicate that to determine the level of micro-RNA in extracellular vesicles, regardless of their cellular origin, the number of particles in the sample should be more than 3125. At the same time, given the high variability of the number of specific micro-RNAs depending on the cellular origin of the vesicles, it is advisable to specify the target threshold of extracellular vesicles during each individual experiment.

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