Abstract
Extracellular vesicles (EVs) are key mediators of intercellular communication. Part of their biological effects can be attributed to the transfer of cargos of diverse types of RNAs, which are promising diagnostic and prognostic biomarkers. EVs found in human biofluids are a valuable source for the development of minimally invasive assays. However, the total transcriptional landscape of EVs is still largely unknown. Here we develop a new method for total transcriptome profiling of plasma-derived EVs by next generation sequencing (NGS) from limited quantities of patient-derived clinical samples, which enables the unbiased characterization of the complete RNA cargo, including both small- and long-RNAs, in a single library preparation step. This approach was applied to RNA extracted from EVs isolated by ultracentrifugation from the plasma of five healthy volunteers. Among the most abundant RNAs identified we found small RNAs such as tRNAs, miRNAs and miscellaneous RNAs, which have largely unknown functions. We also identified protein-coding and long noncoding transcripts, as well as circular RNA species that were also experimentally validated. This method enables, for the first time, the full spectrum of transcriptome data to be obtained from minute patient-derived samples, and will therefore potentially allow the identification of cell-to-cell communication mechanisms and biomarkers.
Highlights
“Liquid biopsies” are being increasingly recognized as transformative in biology and medicine
Blood samples were collected in BD Vacutainer tubes with Acid Citrate Dextrose (ACD) solution, from healthy volunteer individuals participating in the cancer prevention campaign at the A.C. Camargo Cancer Center (ACCCC), after signing an Institutional Review Board-approved written informed consent form
To certify that we were working with bona fide Extracellular vesicles (EVs), the isolated particles were first validated by transmission electron microscopy (TEM), Nanoparticle Tracking Analysis (NTA) and Western blotting
Summary
“Liquid biopsies” are being increasingly recognized as transformative in biology and medicine. Extracellular vesicles (EVs) such as exosomes and microvesicles are involved in a wide variety of physiological processes and have important roles in cell-to-cell communication during development, as well as in health and diseased states[1,2] Their capacity to influence the physiology of the recipient cells/tissues is due to the transfer of their cargo of lipids, proteins, and nucleic acids[3,4], which is produced by their parental cells, selected and loaded into the EVs5, and delivered both locally and to distant sites[6,7]. Recent studies have attempted to profile populations of vesicular RNAs by using generation sequencing (NGS), to allow the identification of a catalogue of vesicle-derived RNAs (Table 1) Most of these studies employed size-selection protocols during NGS library preparation, which has limited the analysis essentially to small RNAs10–13. By not excluding RNAs of any particular size, our method allows an unbiased characterization of the total transcriptional landscape of EVs, which still remains largely unknown
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