Plasma EV Isolation and RNA Analysis

Blood was drawn from each participating veteran in the morning after a night of fasting. Whole blood was collected into 10 mL vacutainer ethylenediaminetetraacetic acid (EDTA) tubes (Becton Dickinson, Franklin Lakes, NJ, USA). Plasma samples were prepared after removing blood cells by centrifugation for 10 min at 1100× g, aliquoted, and stored at −80 until subsequent use. To remove cell debris and platelets, plasma was centrifuged for 10 min at 10,000× g. EVs in plasma samples were isolated using size-exclusion chromatography as previously described [28 (link)]. In brief, 100 μL of plasma was added to the PBS equilibrated column (iZON science, Cambridge, MA, USA) and the sample was fractionated in 500 μL increments with 15 mL of PBS. Fractions 7–9 were combined as the EV fraction, and fractions 12–32 were combined as the depleted fraction. Both the EV and EV-depleted fractions were concentrated using an Amicon Ultra-4 10 K centrifugal filter (EMD Millipore, Billerica, MA, USA). Total RNA, including miRNA, was isolated from all three fractions (whole plasma, EV and EV-depleted) using a modified Qiagen miRNeasy Micro procedure (Qiagen, Germantown, MD, USA). The RNA quality and the concentration was assessed using an RNA Pico assay on an Agilent Bioanalyzer (Santa Clara, CA, USA).