Full-Spectrum Sequencing Analysis of RNA TOP-PCR-Amplified Extracellular Vesicle RNAs (EV-RNAs) for Healthy Males

Extracellular vesicle RNAs (EV-RNAs) possess important biological functions and strong therapeutic potential. Their composition, however, is not yet fully understood. To obtain a comprehensive insight of the composition for healthy individuals, we adopted RNA-based T oligo-primed PCR (RNA TOP-PCR) to amplify all EV-RNA species for three healthy males. The constituent sequences were then sequenced and analyzed. The results showed that rRNAs constitute the major species (31.1–43.6%) of EV-RNAs, followed by ncRNAs (including lncRNAs, 4.3–7.9%, and sncRNAs, 0.4–1.2%), Y RNAs (3.9–9.3%), mRNAs (0.7–1.7%), and tRNAs (~0.1%), together with small amounts of mitochondrial tRNA, pseudogenes, and miRNAs (<0.004% over mappable reads). Noticeably, ~40% of the EV-RNAs remain unannotated, while the annotated EV-mRNAs seem to bear a major mission focusing on signal transduction and oxidative phosphorylation. Motif sequences for lncRNA vesicle localization highly agree with previous reports. Among Y RNAs, RNY3 and RNY4 are the major isoforms. Interestingly, many sequences overlap with precursor miRNAs but do not overlap with mature miRNAs. Taken together, RNA TOP-PCR is a robust method suitable for the study of EV-RNAs complexity, and EV-RNAs-mediated intercellular regulation seems to be much more complicated than previously thought.