The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling

By J. Van Deun, Pieter Mestdagh, R. Sormunen, V. Cocquyt, K. Vermaelen, Jo Vandesompele, M. Bracke, O. De Wever, A. Hendrix. Journal of Extracellular Vesicles, 2014

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Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers.

Subfields: Cancer Research, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/7d0a02464f60798cc677ff451c80f1d552629f3a

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Citations in the corpus

Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics (2020, background): “Furthermore, as has been emphasized by previous studies [37], the structure and biological function of the isolated exosomes can be detrimentally affected by prolonged periods of ultra-centrifugal force, which is very unfavorable for downstream applications such as exosome-based functional studies a”
Mesenchymal Stromal Cells Modulate Macrophages in Clinically Relevant Lung Injury Models by Extracellular Vesicle Mitochondrial Transfer (2017, positive): “It has been shown that different methods of isolation can influence vesicle yield, purity and contents((53)).”
Isolation of Extracellular Vesicles: General Methodologies and Latest Trends (2018, methodology): “In addition, THP is capable of binding calcium ions [147].”
Obstacles and opportunities in the functional analysis of extracellular vesicle RNA – an ISEV position paper (2017, background): “[27,28] This urges the need for including control isolates from non-conditioned culture medium in the RNA analysis.”
Advances in therapeutic applications of extracellular vesicles (2019, methodology): “These precipitation methods have been widely used and demonstrate high recovery of EVs; however, the purity is often rather poor with coprecipitation of nonvesicular-associated protein and nucleic acid contaminants that may confound conclusions of EV content and function (79).”
Isolation of exosomes from whole blood by integrating acoustics and microfluidics (2017, methodology): “Other methods, including immunoaffinity capture (19, 20), precipitation kits such as ExoQuick (System Biosciences) and Total Exosome Isolation (Invitrogen) (12, 21), microfluidics (17, 22, 23), nanoscale lateral displacement arrays (24), nanostructurebased filtration (25), nanoplasmonic chip (26), m”