Comparative analysis of EV isolation procedures for miRNAs detection in serum samples

• DOI: 10.3402/jev.v5.31655
• Publication Date: 2016-06-20T10:40:46Z
• Authors: Zoraida Andreu; Eva Rivas; Aitana Sanguino‐P...; Amalia Lamana; Mónica Marazuela; Isidoro González‐...; Francisco Sánchez...; Hortensia de la F...; María Yáñez‐Mó
• Keywords: Isolation

Extracellular vesicles (EVs) are emerging as potent non-invasive biomarkers. However, current methodologies time consuming and difficult to translate clinical practice. To analyse EV-encapsulated circulating miRNA, we searched for a quick, easy economic method enrich frozen human serum samples EV. We compared the efficiency of several protocols commercial kits isolate EVs. Different methods based on precipitation, columns or filter systems were tested with ultracentrifugation, which is most classical protocol EV assessed purity quantity by nanoparticle tracking analysis western blot cytometry against major protein markers. For biomarker validation, levels set miRNAs determined in fractions their total serum. EVs isolated precipitation-based enriched subgroup that corresponded described be encapsulated into (miR-126, miR-30c miR-143), while detection miR-21, miR-16-5p miR-19a was very low Our results point precipitation using polyethylene glycol (PEG) suitable an cheap enrichment miRNA analyses. The overall performance PEG similar, better than other precipitating reagents, both yield, but comparison them much cheaper. Other presented poorer results, mostly when assessing qPCR Using longitudinal study samples, demonstrated could up 8 years storage. report cut-off value mean fold versus provides estimate degree encapsulation given miRNA.