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Abstract
Extracellular vesicles (EVs), including exosomes and microvesicles, have been shown to carry a variety of biomacromolecules including mRNA, microRNA and other non-coding RNAs. Within the past 5 years, EVs have emerged as a promising minimally invasive novel source of material for molecular diagnostics. Although EVs can be easily identified and collected from biological fluids, further research and proper validation is needed in order for them to be useful in the clinical setting. In addition, innovative and more efficient means of nucleic acid profiling are needed to facilitate investigations into the cellular and molecular mechanisms of EV function and to establish their potential as useful clinical biomarkers and therapeutic tools. In this article, we provide an overview of recent technological improvements in both upstream EV isolation and downstream analytical technologies, including digital PCR and next generation sequencing, highlighting future prospects for EV-based molecular diagnostics.
Acknowledgements
The authors thank S Ghosh, M Lackner, D Taylor and AJ Trachtenberg for critical reading of this manuscript. The authors apologize to colleagues whose primary papers were not cited due to space constraints. The views or opinions presented in this article are solely those of the authors and do not necessarily represent the official views of their employers.
Financial & competing interests disclosure
S Jia is an employee of Genentech Inc., and owns stock in Roche Holdings. Johan Skog is an employee and share holder of Exosome Diagnostics Inc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.
No writing assistance was utilized in the production of this manuscript.
Key issues
Extracellular vesicles (EVs) are membranous, cell-derived, mixed populations of vesicles, ranging from approximately 40–5000 nm in diameter, and are released by a variety of cells into the intercellular microenvironment and various extracellular biofluids.
EVs are released by a variety of cell types into their microenvironments, and are detectable in biofluids such as blood, breast milk, urine, cerebrospinal fluid, saliva, amniotic fluid and ascites fluid.
EVs play roles in pathogenesis of different pathological conditions and can also affect other cells by transfer of lipids, genetic cargo, transfer of receptors and other proteins and ultimately by triggering activation of important pathways.
EVs, including exosomes and microvesicles, have been shown to carry a variety of biomacromolecules and nucleic acids including lipid, protein, DNA and RNA components, and they may be used for biomarker discovery and disease monitoring.
The majority of EV isolation approaches are based on their physical and chemical properties such as size, density and solubility and vary in yield, purity and integrity of the final preparation.
Several technology platforms have been successfully used in EV RNA profiling including microarrays, quantitative PCR, digital PCR and next-generation sequencing.
A critical problem in EV research has been poor reproducibility and the lack of standard isolation methods, standard biomarkers or standard assay methods.
Several EV RNA-based diagnostic companies are working with clinical laboratories to develop, validate and commercialize EV RNA-based assays.