title: KeepEX, a simple dilution protocol for improving extracellular vesicle yields from urine.
abstract: Urinary extracellular vesicles (EVs) are a promising source of biomarkers, which can be obtained in a non-invasive manner. However, the yield of EVs from urine samples may be insufficient for various analyses due to the entrapment of EVs by the Tamm-Horsfall protein (THP) meshwork. Here, we developed a simple dilution protocol to increase the urinary EV yield by disrupting the interaction between THP filaments and EVs with the help of alkaline pH and lowered ionic concentration. The integrity of the EVs and THP was assessed by electron microscopy. The effect of the protocol on the EV yield was quantified against an undiluted control by western blotting of four EV markers, nanoparticle tracking analysis and measuring of the RNA/miRNA concentration of the EV samples. The average EV yield from the dilution protocol was 2-7 fold the yield from the undiluted control i.e. increased by 130-624% as measured by western blotting and NTA. The yield increased most from samples with a high THP to EV ratio. The morphology and size range of the EVs were unaltered by the protocol. However, RNA/miRNA yields were the same as from the undiluted control and THP filaments could still be detected in EV samples. The dilution protocol, that we named KeepEX, provides a simple and efficient way to prevent loss of EVs thus increasing their yield from urine. Since KeepEX does not require individual adjustment of sample pH nor extra centrifugation steps, it could be used on its own or in combination with other EV purification protocols to improve EV isolation particularly from small urine volumes.
authors: Institute for Molecular Medicine Finland (FIMM), Helsinki Urological Biobank, University of Helsinki, Finland. Electronic address: Maija.Puhka@Helsinki.fi.
authors: M
authors: M
authors: Puhka
authors: Institute for Molecular Medicine Finland (FIMM), Helsinki Urological Biobank, University of Helsinki, Finland.
authors: M-E
authors: ME
authors: Nordberg
authors: EV Group, Division of Biochemistry and Biotechnology, Department of Biosciences and Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Finland; Finnish Red Cross Blood Service, Helsinki, Finland.
authors: S
authors: S
authors: Valkonen
authors: Institute for Molecular Medicine Finland (FIMM), Helsinki Urological Biobank, University of Helsinki, Finland; Helsinki University Central Hospital, Department of Urology, Helsinki, Finland.
authors: A
authors: A
authors: Rannikko
authors: Institute for Molecular Medicine Finland (FIMM), Helsinki Urological Biobank, University of Helsinki, Finland.
authors: O
authors: O
authors: Kallioniemi
authors: EV Group, Division of Biochemistry and Biotechnology, Department of Biosciences and Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Finland.
authors: P
authors: P
authors: Siljander
authors: Institute for Molecular Medicine Finland (FIMM), Helsinki Urological Biobank, University of Helsinki, Finland; Orion Corporation, Orion Pharma, Orionintie 1, P.O. Box 65, 02101 Espoo, Finland.
authors: T M
authors: TM
authors: Af Hällström
mesh_terms: D002498:Centrifugation; D000067128:Extracellular Vesicles / Q000648:ultrastructure; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046529:Microscopy, Electron, Transmission; D011506:Proteins / Q000032:analysis; D014325:Tromethamine / Q000737:chemistry; D014556:Urine / Q000166:cytology*
chemical_list: D011506:Proteins; D014325:Tromethamine
keywords: Exosomes; Extracellular vesicles; THP; Urine; Yield