Gentle, Effective, and Timely Isolation of Extracellular Vesicles that is Amenable for Transcriptomics Using Next-Generation Aqueous Two-Phase System Technology

The isolation of extracellular vesicles (EVs) using currently available methods frequently compromises purity and yield to prioritize speed. Here, we present a next generation aqueous two-phase system (ATPS) for isolation of EVs regardless of scale and volume and is superior to conventional methods such as ultracentrifugation and commercial kits. This is made possible by the two aqueous phases, one rich in polyethylene glycol (PEG), and the other rich in dextran (DEX), whereby fully encapsulated lipid vesicles preferentially migrate to the DEX-rich phase to achieve a local energy minimum for the EVs. Isolated EVs as found in the DEX-rich phase are more amenable to biomarker analysis such as nanoscale flow cytometry when using various pre-conjugated antibodies specific for CD9, CD63, and CD81. TRIzol RNA isolation is further enabled by the addition of Dextranase, a critical component of this next generation ATPS method. This negates the use of specialized EV RNA extraction kits. The use of Dextranase also enables more accurate immunoreactivity of pre-conjugated antibodies for detection of EVs by nanoscale flow cytometry. Transcriptomics of isolated EVs with ATPS revealed strong overlap between micro RNA (miRNA), circular RNA (circRNA), and small nucleolar RNA (snoRNA) profiles of EVs isolated compared to UC and was superior to other kits. Overall, this ATPS method stands out as a rapid and highly effective approach to isolate EVs, ensuring optimal extraction and analysis of nucleic acids. Overall design: Media was collected from various cells and extracelluar vesicles (Evs) were isolated using either ultracentrifuge, the aqueous two-phase system (ATPS), or Qiagen exoRNeasy Midi Kit. Total RNA was extracted using Trizol and small RNA library was prepared and sequenced.