Plasma and EV RNA Extraction

Total RNA was isolated from plasma and EV samples using TRIzol LS^TM Reagent (Thermo Fisher Scientific, Waltham, MA, USA) as per manufacturer’s instructions with some modifications. Briefly, 750 µL TRIzol LS was added to 200 µL of plasma and 100 µL of EVs (diluted up to 200 µL in RNase-free water). Samples were spiked with 2.5 femtomoles of cel-miR-54 mirVana mimic (MC10279, Thermo Fisher Scientific, Waltham, MA, USA) to normalise for technical variability in RNA extraction and RT-qPCR efficiency. This exogenous control was employed in the absence of established endogenous genes for normalisation of plasma or EV-derived miRNA RT-qPCR data [24 (link),25 (link)]. Isopropyl RNA precipitation was facilitated by addition of 40 µg of RNase-free glycogen (Thermo Fisher Scientific, Waltham, MA, USA), and the RNA pellet was washed with ice cold 80% ethanol. RNA was resuspended in 30 µL of RNase-free water.