Isolation, Extraction and Deep-Sequencing Analysis of Extracellular RNAs (exRNAs) from Human Plasma

Extracellular RNAs (exRNAs) are secreted by nearly all cell types and are now known to play multiple physiological roles. Human plasma, a readily available sample for biomedical analysis, was reported to contain various subpopulations of exRNA, some of which are most likely components of plasma ribonucleoproteins (RNPs), while others are encapsulated into extracellular vesicles (EVs) of different size, origin, and composition. Unbiased analysis of exRNA composition can be performed with prefractionation of plasma exRNA followed by library preparation, sequencing, and bioinformatics analysis. In addition to "mature," adaptor ligation-competent RNA species (5'-P/3'-OH), human plasma contains a substantial proportion of degraded RNA fragments, featuring 5'-OH/3'-P or cyclophosphate extremities, which can be made competent for ligation using appropriate treatment. Polyethylene glycol (PEG)-based precipitation kits for EV isolation yield a fraction that is highly contaminated by large RNPs and EV-associated RNAs. Purer EV preparations are obtained by using Proteinase K and RNase A treatment, as well as by size-exclusion chromatography (SEC).