Diversity and heterogeneity of extracellular RNA in human plasma

Extracellular RNAs (exRNAs) are secreted by nearly all cell types and are now known to play multiple physiological roles. In humans, exRNA populations are found in nearly any physiological liquid and are attracting growing interest as a potential source for biomarker discovery. Human plasma, a readily available sample for biomedical analysis, 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. This variation explains the extreme complexity of the human exRNA fraction in plasma. In this work, we aimed to characterize exRNA species from blood samples of healthy human donors to achieve the most comprehensive overview of the species, sizes and origins of the exRNA present in plasma fractions. Unbiased analysis of exRNA composition was performed with prefractionation of plasma exRNA followed by library preparation, sequencing and bioinformatics analysis. Our results demonstrate that, in addition to “mature”, adaptor ligation-competent RNA species (5′-P/3′-OH), human plasma contains a substantial proportion of degraded RNA fragments (5′-OH/3′-P or cycloP), which can be made competent for ligation using appropriate treatments. These degraded RNAs represent the major fraction in the overall population and mostly correspond to rRNA, in contrast to mature products, which mostly contain miRNAs and hY4 RNA fragments. Precipitation polyethylene glycol (PEG)-based kits for EV isolation yield a fraction that is highly contaminated by large RNPs and by RNA loosely bound to EVs. Purer EV preparations are obtained by using proteinase K and RNase A treatment, as well as by size-exclusion chromatography (SEC). These samples have rather distinct RNA compositions compared to PEG-precipitated EV preparations and contain a substantial proportion of exRNA of non-human origin, arising from human skin and gut microbiota, including viral microbiota. These exogenous exRNAs represent up to 75–80% of total RNA reads in highly purified extracellular vesicles, paving the way for biomedical exploitation of these non-human biomarkers.