Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing

MicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies miRNA patterns their validation still frequent due to differences sample origin, EV isolation, sequencing methods. The aim of the present study is find a reliable isolation method for sequencing, adequate clinical application. To this aim, two comparative studies were performed parallel with same human plasma sample: (i) characterization EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), its combination (SEC+GRAD) (ii) evaluation yield sequences NextSeq 500 (Illumina) library preparation protocols: NEBNext, NEXTFlex, SMARTer smRNA-seq. conclusion comparison that recovery largest amount reproducibility attained SEC, but GRAD SEC+GRAD yielded purer preparations. NEBNext showed highest number miRNAs recovered diversity miRNAs. These results render retrieval as applications samples.