Extracellular Vesicle Isolation and Analysis

Extracellular vesicles were precipitated from 1ml serum using an isolation kit (miRCURY Exosome Isolation Kit, Qiagen, Venlo, Netherlands) as previously described (19 (link)) followed by extraction of cell-free RNA. For qPCR, an EV isolation and RNA extraction were performed from a second 1 ml serum aliquot. Particle concentration and size distribution were evaluated by Nanoparticle Tracking Analysis on a ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and visualized by transmission electron microscopy. Following the Shapiro-Wilk normality test, statistical significance of particle tracking analysis data were evaluated by ordinary one-way ANOVA followed by Tukey’s multiple comparison test using Graphpad Prism (version 8.3.1) with a significance level of p ≤ 0.05. Particle concentration and size were reported as mean values ± standard deviation. Additionally, precipitated vesicles were visualized after negative staining with 4% uranyl acetate by transmission electron microscopy using a Zeiss EM900 (Carl Zeiss Microscopy GmbH, Jena, Germany) with a wide-angle dual speed 2KCCD camera at 80 kV.