Rna 6000 nano and pico total rna kits

Total RNA from cells and isolated EVs was extracted using Tri‐Reagent (Sigma–Aldrich, Dorset, UK), as per the manufacturer's instructions. RNA samples were eluted in 11 µl of RNAse‐free water, aliquoted, and stored at –80°C. The size profile of isolated RNA was measured by capillary electrophoresis, using RNA 6000 Nano and Pico total RNA kits (Agilent Technologies UK Ltd, Cheshire, UK), in an Agilent Bioanalyzer 2100 instrument (Agilent Technologies UK Ltd). To assess purity of EV‐associated RNA, isolated EVs were incubated with proteinase K (0.05 µg/µl; Sigma–Aldrich) for 10 min at 37°C. proteinase K activity was inhibited by the addition of PMSF (5 mM; Sigma–Aldrich) for 10  min at room temperature, followed by further incubation at 90°C for 5 min. EVs were then incubated with RNase A (0.5 µg/µl) for 20 min at 37°C to degrade unprotected RNA. For Control samples, PBS was added in place of proteinase K, PMSF, and RNase A. Prior to further analysis, RNA concentration was assessed by NanoDrop™ 2000 spectrophotometer (ThermoFisher Scientific), as per the manufacturer's instructions, and used to normalise sample input.

Total RNA from cells and EV subpopulations was isolated using the miRNAeasy micro kit (Qiagen, Hilden, Germany), according to the manufacturer´s instructions. RNA samples were eluted in 14 μL of RNAse-free water, aliquoted, and stored at -80 °C. The quality and sized of the isolated RNA was measured by capillary electrophoresis using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). RNA from each sample was denatured at 72 °C for 2 min and loaded into RNA 6000 Nano and Pico total RNA kits (Agilent Technologies) to analyze RNA profile and concentration. The RNA concentration was used as a normalizing loading factor for all EV samples.