design: Whole peripheral blood was collected in EDTA tubes and processed within two hours of drawing. For plasma isolation for samples from prostate cancer and a group of healthy donors, blood was spun at 900g for 7 min, the top fraction collected, and then, centrifuged again for 10 min at 2500g. For the isolation of plasma from patients with low-grade-glioma and a group of healthy volunteers, blood was spun at 120g for 20 minutes, then for 360 for additional 20 min and the processing was completed with a last step of centrifugation at 4800g for 15 min. The resulting platelet-poor plasma was then aliquoted and stored at -80C until processing. All plasma samples from patients with tumors were compared to samples from healthy donors processed with the same protocol. For the isolation of EVs from plasma we used size exclusion chromatography (SEC) following the protocol of Bing et al1. For that purpose, 1ml of plasma was diluted with 500l of PBS and loaded to a pre-packed column. Ten fractions of 0,5ml were collected per sample using the automatic fraction collector from iZon on prepacked columns (qEVoriginal). EV-enriched fractions 9 and 10 were then split into 250 l aliquots and 5 parts of Qiazol were added per aliquot. Samples were left at room temperature for 20minutes and then frozen at -80C for a minimum of 12h. RNA isolation was performed using miRNeasy Serum/Plasma kit from Qiagen adjusting the volumes of the reagents to the volume of all combined fractions. The final elution volume was 15 l. RNA was stored at -80C prior submission to GenomeScan for library preparation and sequencing. Libraries were prepared using NEBNext Small RNA library Prep Seq for Illumina using 15 l for library preparation and then adjusted to equal concentrations after qPCR. Sequence read length was set at 50bp, single-end and with coverage of 20106 reads per sample. Samples were finally demultiplexed.