Extracting RNA from Urinary Extracellular Vesicles

To extract RNA from uEVs isolated by ultracentrifugation, we employed miRCURY™ RNA Isolation Kit Cell & Plant (Exiqon). In average, 1.5e⁷ vesicles were used per retrotranscription reaction. In addition, a set of samples was extracted by Norgen Biotek Exosomal RNA purification kit, following the manufacturers' instructions. For cell lines, RNA was extracted using NucleoSpin^® RNA isolation kit from Macherey-Nagel (ref: 740955.240C). cDNA was synthesized from 0.1–1 μg of RNA using Superscript III (Life Technologies) following the manufacturer's recommendations. For prostate tissue samples, RNA was extracted as reported in [16 ]. Quantitative Real Time PCR (Taqman qRTPCR) was performed as previously described [18 (link)]. Universal Probe Library (Roche) primers and probes employed are detailed in Supplementary Table S2. β-ACTIN (Hs99999903_m1) and GAPDH (Hs02758991_g1) housekeeping assays were from Applied Biosystems and showed similar results.