Advanced search
1 file | 1.91 MB

Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research

Glenn Vergauwen (UGent), Bert Dhondt (UGent), Jan Van Deun (UGent), Eva De Smedt (UGent), Geert Berx (UGent), Evy Timmerman (UGent), Kris Gevaert (UGent), Ilkka Miinalainen, Veronique Cocquyt (UGent), Geert Braems (UGent), et al.
Author
Organization
Abstract
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.
Keywords
URINARY EXOSOMES, PLASMA, CANCER, SUBPOPULATIONS, MICROPARTICLES, IMPACT

Downloads

  • s41598-017-02599-y.pdf
    • full text
    • |
    • open access
    • |
    • PDF
    • |
    • 1.91 MB

Citation

Please use this url to cite or link to this publication:

Chicago
Vergauwen, Glenn, Bert Dhondt, Jan Van Deun, Eva De Smedt, Geert Berx, Evy Timmerman, Kris Gevaert, et al. 2017. “Confounding Factors of Ultrafiltration and Protein Analysis in Extracellular Vesicle Research.” Scientific Reports 7.
APA
Vergauwen, G., Dhondt, B., Van Deun, J., De Smedt, E., Berx, G., Timmerman, E., Gevaert, K., et al. (2017). Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research. SCIENTIFIC REPORTS, 7.
Vancouver
1.
Vergauwen G, Dhondt B, Van Deun J, De Smedt E, Berx G, Timmerman E, et al. Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research. SCIENTIFIC REPORTS. 2017;7.
MLA
Vergauwen, Glenn, Bert Dhondt, Jan Van Deun, et al. “Confounding Factors of Ultrafiltration and Protein Analysis in Extracellular Vesicle Research.” SCIENTIFIC REPORTS 7 (2017): n. pag. Print.
@article{8522427,
  abstract     = {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.},
  articleno    = {2704},
  author       = {Vergauwen, Glenn and Dhondt, Bert and Van Deun, Jan and De Smedt, Eva and Berx, Geert and Timmerman, Evy and Gevaert, Kris and Miinalainen, Ilkka and Cocquyt, Veronique and Braems, Geert and Van den Broecke, Rudy and Denys, Hannelore and De Wever, Olivier and Hendrix, An},
  issn         = {2045-2322},
  journal      = {SCIENTIFIC REPORTS},
  keyword      = {URINARY EXOSOMES,PLASMA,CANCER,SUBPOPULATIONS,MICROPARTICLES,IMPACT},
  language     = {eng},
  pages        = {12},
  title        = {Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research},
  url          = {http://dx.doi.org/10.1038/s41598-017-02599-y},
  volume       = {7},
  year         = {2017},
}

Altmetric
View in Altmetric
Web of Science
Times cited: