Exorneasy serum plasma

EVs were isolated using the exoRNeasy Serum/Plasma (Qiagen cat# 77044) assay, followed by RNA isolation using the miRNeasy serum/plasma advanced kit (Qiagen cat# 217204), as per manufacturer’s directions. Briefly, thrombin was added to the plasma, incubated for 5 minutes at room temperature, and centrifuged at 2,500g for 15 min. The supernatant was then mixed with precipitation buffer and incubated for 60 min at 4°C. Following centrifugation at 13,000g for 5 min, the pellet was resuspended and served for RNA isolation. The resuspended pellet was lysed, protein was precipitated and removed, isopropanol was added to the supernatant, and the sample loaded onto the column. Following three washes, RNA was eluted and stored at −80°C.
EVs were characterized post-isolation based on transmission electron microscopy and expression of EV markers (CD9, CD63, CD81) by Western blot, as previously published (10 (link)), and their concentrations determined using nanoparticle tracking analysis. In addition, their cargo was compared to typical EV markers listed in ExoCarta (http://www.exocarta.org), a web-based resource of exosomal cargo.

Using the exoRNeasy Serum/Plasma (Qiagen cat# 77044) assay, circulating EVs were isolated following the vendor’s instructions, and RNA was subsequently isolated with the miRNeasy serum/plasma advanced kit (Qiagen cat# 217204). Initially, thrombin was added to the plasma for 5 min, and the sample was centrifuged at 2,500×g for 15 min. After being mixed with precipitation buffer and incubated at 4°C for 60 min, the supernatant was centrifuged at 13,000×g for 5 min. The pellet was resuspended and lysed, and proteins were precipitated and removed. Isopropanol was added to the supernatant, which was then loaded onto the column. RNA was eluted following three washes and stored at −80°C.