title: The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling.
abstract: 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.
authors.affiliation: Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Van Deun
authors.affiliation: Center for Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Mestdagh
authors.affiliation: Biocenter Oulu, Department of Pathology, Oulu University Hospital, University of Oulu, Oulu, Finland.
authors.forename: Raija
authors.initials: R
authors.lastname: Sormunen
authors.affiliation: Department of Medical Oncology, Ghent University Hospital, Ghent, Belgium.
authors.forename: Veronique
authors.initials: V
authors.lastname: Cocquyt
authors.affiliation: Department of Internal Medicine, Ghent University Hospital, Ghent, Belgium.
authors.forename: Karim
authors.initials: K
authors.lastname: Vermaelen
authors.affiliation: Center for Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
authors.affiliation: Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium.
authors.forename: Marc
authors.initials: M
authors.lastname: Bracke
authors.affiliation: Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium.
authors.forename: Olivier
authors.initials: O
authors.lastname: De Wever
authors.affiliation: Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium.
authors.forename: An
authors.initials: A
authors.lastname: Hendrix
keywords: ExoQuick; OptiPrep; exosomes; extracellular vesicles; omics; ultracentrifugation