Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research |
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Author: | Vergauwen, Glenn1,2,3; Dhondt, Bert1,4,3; Van Deun, Jan1,3; |
Organizations: |
1Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University, Ghent, Belgium 2Department of Gynaecology, Ghent University Hospital, Ghent, Belgium 3Cancer Research Institute Ghent, Ghent, Belgium
4Department of Urology, Ghent University Hospital, Ghent, Belgium
5Molecular and Cellular Oncology Lab, Department for Molecular Biomedical Research, Ghent, Belgium 6VIB Medical Biotechnology Center, VIB, Ghent University, A. Baertsoenkaai 3, Ghent, Belgium 7Department of Biochemistry, Ghent University, A. Baertsoenkaai 3, Ghent, Belgium 8Biocenter Oulu, Department of Pathology, Oulu University Hospital, University of Oulu, Oulu, Finland 9Department of Medical Oncology, Ghent University Hospital, Ghent, Belgium |
Format: | article |
Version: | published version |
Access: | open |
Online Access: | PDF Full Text (PDF, 1.7 MB) |
Persistent link: | http://urn.fi/urn:nbn:fi-fe201708228178 |
Language: | English |
Published: |
Springer Nature,
2017
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Publish Date: | 2017-08-22 |
Description: |
AbstractIdentification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications. see all
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Series: |
Scientific reports |
ISSN: | 2045-2322 |
ISSN-E: | 2045-2322 |
ISSN-L: | 2045-2322 |
Volume: | 7 |
Article number: | 2704 |
DOI: | 10.1038/s41598-017-02599-y |
OADOI: | https://oadoi.org/10.1038/s41598-017-02599-y |
Type of Publication: |
A1 Journal article – refereed |
Field of Science: |
1182 Biochemistry, cell and molecular biology |
Subjects: | |
Funding: |
The work was supported by Fund for Scientific Spearheads of the Ghent University Hospital, Concerted Research Actions from Ghent University (GOA-01GB1013W), Kom Op Tegen Kanker, the National Cancer Plan (KPC_29_012), Krediet Aan Navorsers and PhD (JVD and EDS) and post-doctoral position (AH) from Fund for Scientific Research Flanders (FWO). |
Copyright information: |
© The Author(s) 2017. Open Access
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https://creativecommons.org/licenses/by/4.0/ |