Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research
Vergauwen, Glenn; Dhondt, Bert; Van Deun, Jan; De Smedt, Eva; Berx, Geert; Timmerman, Evy; Gevaert, Kris; Miinalainen, Ilkka; Cocquyt, Véronique; Braems, Geert; Van den Broecke, Rudy; Denys, Hannelore; De Wever, Olivier; Hendrix, An (2017-06-02)
Vergauwen, G., Dhondt, B., Van Deun, J., De Smedt, E., Berx, G., Timmerman, E., Gevaert, K., Miinalainen, I., Cocquyt, V., Braems, G., Van den Broecke, R., Denys, H., De Wever, O., Hendrix, A. (2017) Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research. Scientific Reports, 7 (1), 2704. doi:10.1038/s41598-017-02599-y
© The Author(s) 2017. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ .
Identification 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.
- Avoin saatavuus