Nuclease (RNase) protection assay.

Purified SE pellets derived from 1 ml of seminal plasma were resuspended in 200 μl of nuclease-free 1× PBS (pH 7.4; Ambion, Thermo Fisher Scientific) and divided into four equal fractions, each receiving one of the following treatments: (i) no treatment, (ii) RNase treatment only, (iii) protease followed by RNase treatment, and (iv) detergent followed by protease and RNase treatment. Detergent treatment was performed with 1% NP-40 (Thermo Fisher Scientific) for 15 min on ice. Fractions subjected to protease treatment were incubated with pronase (600 μg/ml; Roche) for 20 min at 40°C, followed by enzyme inactivation at 80°C for 15 min. Finally, fractions subjected to RNase treatment were incubated for 30 min at 37°C with Ambion RNase cocktail enzyme mix (final concentrations of 12 U/ml of RNase A and 500 U/ml of RNase T1; Thermo Fisher Scientific). Following treatment, exosomal RNA was isolated as indicated above, and its yield and size distribution were assessed using an Agilent RNA 6000 Pico kit (Agilent Technologies, Inc.). The percentage of protected RNA was estimated after three treatment replicates.