Extracellular vesicles, RNA sequencing, and bioinformatic analyses: Challenges, solutions, and recommendations.

Extracellular vesicles (EVs) are heterogeneous entities secreted by cells into their microenvironment and systemic circulation. Circulating EVs carry functional small RNAs and other molecular footprints from their cell of origin, and thus have evident applications in liquid biopsy, therapeutics, and intercellular communication. Yet, the complete transcriptomic landscape of EVs is poorly characterized due to critical limitations including variable protocols used for EV-RNA extraction, quality control, cDNA library preparation, sequencing technologies, and bioinformatic analyses.