Abstract
Exosomes and other extracellular vesicles (EVs) are considered the main vehicles transporting RNAs in extracellular samples, including human bodily fluids. However, a major proportion of extracellular RNAs (exRNAs) do not copurify with EVs and remain in ultracentrifugation supernatants of cell-conditioned medium or blood serum. We have observed that nonvesicular exRNA profiles are highly biased toward those RNAs with intrinsic resistance to extracellular ribonucleases. These highly resistant exRNAs are interesting from a biomarker point of view, but are not representative of the actual bulk of RNAs released to the extracellular space. In order to understand exRNA dynamics and capture both stable and unstable RNAs, we developed a method based on size-exclusion chromatography (SEC) fractionation of RNase inhibitor (RI)-treated cell-conditioned medium (RI-SEC-seq). This method has allowed us to identify and study extracellular ribosomes and tRNAs, and offers a dynamical view of the extracellular RNAome which can impact biomarker discovery in the near future. Graphical abstract: Overview of the RI-SEC-seq protocol: sequencing of size-exclusion chromatography fractions from nonvesicular extracellular samples treated or not with RNase inhibitors (+/- RI).
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