Isolation and RNA Extraction from PBMCs and Plasma

PBMCs were isolated from 6 ml of peripheral blood in ethylenediaminetetraacetic acid dipotassium salt dihydrate (K₂-EDTA) anticoagulant tubes by density gradient centrifugation at 1200×g for 10 min at room temperature with brake using Ficoll Histopaque Plus (Cytiva Sweden AB, cat# 17144003, lot #10308253) and SepMate-15 tube (STEMCELL Technologies, Canada, cat# 85415, lot#327374). Isolation of total RNA from PBMCs was performed using TRIzol RNA Isolation Reagent (Life Technologies, US, cat# 15596018, lot#19698001) and RNA concentration and purity were evaluated by NanoDrop One (Thermo Fisher Scientific Inc., US). Platelet-poor plasma was collected in K₂-EDTA-anticoagulant tubes (BD Vacutainer, cat# 368860, lot# 2203608) and isolated as previously reported [19 (link)]. Briefly, cell- and platelet-free plasma was prepared following a 2-steps centrifugation protocol: samples were initially centrifuged at 1500g for 15ʹ at 4 °C. The supernatant was collected and centrifuged again at 14,000g for 15ʹ at 4 °C and plasma aliquots were stored at − 80 °C. RNA was isolated using NucleoSpin miRNA Plasma (cat# 740981.50, lot# 2105/001, Macherey–Nagel, Germany) according to the manufacturer’s instructions.