Isolation and Purification of Urinary Exosomes

Exosome preparation was performed with the Urine Exosome Purification and RNA Isolation Midi Kit (Norgen, Thorold, ON, Canada) according to manufacturer’s instructions with minor modifications. The centrifugation steps were performed as described in the urine processing part, to ensure cell- and debris-free urine. After elution of the exosome fraction, the exosome solution was treated with 0.0125 U/μl of the RNase cocktail RiboShredder (Epicentre, Madison, WI, USA) for 30 min at 37°C, to eliminate cell-free non-exosomal RNA. After RNase treatment, the exosome solution was stored on ice and Lysis Buffer A plus Lysis Additive B were added immediately. RNA was eluted in 75 μl Elution buffer. The miRNA concentrations were determined with the Qubit miRNA-Assay and the Qubit 2.0 (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer`s instructions using 15 μl sample volume.