Syn cel mir 39 3p miscript mirna mimic

RNA was extracted using miRNeasy Serum/Plasma Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Briefly, 250 µL of serum, plasma or hemolysis controls were thawed on ice and centrifuged at 16,000 g and at 4°C for 10 minutes to remove cellular debris. Thereafter 1000 µL of QIAzol Lysis Reagent was added to 200 µL of supernatant. After incubation for 5 minutes, 25 fmol of synthetic cel-miR-39 (Syn-cel-miR-39-3p miScript miRNA Mimic, QIAGEN) was spiked in. Total RNA including small RNA was extracted using QIAcube (QIAGEN) and eluted in 30 µL of RNase-free water. RNA was stored at −80°C until further usage.

Total Exosome Isolation reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used for EV purification from serum. The miRNeasy Mini Kit and Syn-cel-miR-39-3p miScript miRNA Mimic for serum EV RNA purification was purchased from QIAGEN (Hilden, Germany). The following antibodies were used for ExoScreen detection of EVs; biotin-conjugated anti-mouse CD9 (clone MZ3, Biolegend, San Diego, CA, USA), anti-mouse CD9 (clone MZ3, Biolegend), and anti-human CD9 (clone 12A12, Shionogi & Co., LTD, Osaka, Japan). ChromaLink™ Biotin Labelling Kit (Solulink, Inc., San Diego, CA, USA) was used for biotinylation of anti-human CD9 antibody. AlphaLISA reagents (PerkinElmer, Waltham, MA, USA) for ExoScreen included AlphaScreen Streptavidin Donor Beads, unconjugated AlphaLISA Acceptor Beads and AlphaLISA Universal Buffer. Conjugation of AlphaLISA Acceptor Beads to anti-human or -mouse CD9 antibodies was performed according to the manufacturer’s protocol.