Exosome Isolation from Plasma via ExoQuick

Exosomes were isolated using the ExoQuick Exosome Precipitation Solution (SBI, Mountain View, CA) as previously described^{14 (link)}. Briefly, the plasma was thawed on ice and centrifuged at 3000 g for 10 min to remove possible residual cell debris. After incubated with thromboplastin D (Thermo, Middletown, CA) at 37 °C for 15 min and centrifuged, the 250 μL supernatant was aspirated to a new tube and mixed with 65 μL ExoQuick Solution. To digest free RNAs outside of exosomes, RNaseA (Sigma, St. Louis, MO) was added to the mixture at final concentration of 10 µg/ml. After keeping the mixture at 4 °C overnight, Murine RNase inhibitor (NEB, Ipswich, MA) was added at 150 units/ml before the precipitation of exosomes by centrifuging at 1500 g for 30 min. The exosome pellet was dissolved in 50 µL PBS and subjected to RNA extraction immediately.