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Purification of High Yield Extracellular Vesicle Preparations Away from Virus
Published on: September 12, 2019
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KeepEX, a simple dilution protocol for improving extracellular vesicle yields from urine
M Puhka1, M-E Nordberg1, S Valkonen2
1Institute for Molecular Medicine Finland (FIMM), Helsinki Urological Biobank, University of Helsinki, Finland.
Summary
A new KeepEX dilution protocol significantly boosts urinary extracellular vesicle (EV) yield by up to 7-fold. This method enhances biomarker discovery by preventing EV loss caused by Tamm-Horsfall protein interactions.
Area of Science:
- Biomarker Discovery
- Extracellular Vesicle Research
- Urine Proteomics
Background:
- Urinary extracellular vesicles (EVs) are valuable non-invasive biomarkers.
- EV yield is often limited by entrapment within the Tamm-Horsfall protein (THP) meshwork.
- Insufficient EV yield hinders comprehensive urinary biomarker analysis.
Purpose of the Study:
- To develop a simple protocol to enhance urinary EV yield.
- To overcome EV loss due to THP entrapment.
- To improve the efficiency of EV isolation from urine samples.
Main Methods:
- A dilution protocol (KeepEX) was developed using alkaline pH and lowered ionic concentration.
- EV integrity and THP interaction were assessed via electron microscopy.
- EV yield was quantified using western blotting, nanoparticle tracking analysis (NTA), and RNA/miRNA concentration measurements, compared to an undiluted control.
Main Results:
- The KeepEX protocol increased EV yield by 2-7 fold (130-624%) compared to the undiluted control.
- Yield improvement was most significant in samples with high THP to EV ratios.
- EV morphology and size remained unaltered; RNA/miRNA yields were comparable, and THP filaments were still detectable.
Conclusions:
- The KeepEX protocol effectively increases urinary EV yield by disrupting THP-EV interactions.
- This simple, efficient method enhances EV isolation without altering EV integrity or requiring complex steps.
- KeepEX offers a valuable approach to improve EV biomarker recovery, especially from small urine volumes.
