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J Extracell Vesicles. 2014 Sep 18;3. doi: 10.3402/jev.v3.24858. eCollection 2014.

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

Author information

1
Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium.
2
Center for Medical Genetics, Ghent University, Ghent, Belgium.
3
Biocenter Oulu, Department of Pathology, Oulu University Hospital, University of Oulu, Oulu, Finland.
4
Department of Medical Oncology, Ghent University Hospital, Ghent, Belgium.
5
Department of Internal Medicine, Ghent University Hospital, Ghent, Belgium.

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.

KEYWORDS:

ExoQuick; OptiPrep; exosomes; extracellular vesicles; omics; ultracentrifugation

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