Polyethylene glycol improves current methods for circulating extracellular vesicle-derived DNA isolation

BACKGROUND: Extracellular vesicles (EVs) are small membrane-bound vesicles which play an important role in cell-to-cell communication. Their molecular cargo analysis is presented as a new source for biomarker detection, and it might provide an alternative to traditional solid biopsies. However, the most effective approach for EV isolation is not yet well established. RESULTS: Here, we study the efficiency of the most common EV isolation methods-ultracentrifugation, Polyethlyene glycol and two commercial kits, Exoquick® and PureExo®. We isolated circulating EVs from the bloodstream of healthy donors, characterized the size and yield of EVs and analyzed their protein profiles and concentration. Moreover, we have used for the first time Digital-PCR to identify and detect specific gDNA sequences, which has several implications for diagnostic and monitoring many types of diseases. CONCLUSIONS: Our findings present Polyethylene glycol precipitation as the most feasible and less cost-consuming EV isolation technique.We are grateful for the financial support from the ‘Fondo de Investigaciones Sanitarias’ (FIS) (PI14_00077), the Miguel Servet Program (CP11/00147) del Instituto de Salud Carlos III (AAS), and the Ministerio de Economía y Competi‑tividad–FEDERER (RTC‑2016‑4990‑1, RTC‑2015‑3846‑1). SER was supported by FPI‑CEU predoctoral fellowship.S