title: Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples.
abstract: Urine sample analysis is irreplaceable as a non-invasive method for disease diagnosis and follow-up. However, in urine samples, non-degraded protein and RNA may be only found in urinary extracellular vesicles (uEVs). In recent years, various methods of uEV enrichment using low volumes of urine and unsophisticated equipment have been developed, with variable success. We compared the results of the differential ultracentrifugation procedure with 4 of these methods. The methods tested were a lectin-based purification, Exoquick (System Biosciences), Total Exosome Isolation from Invitrogen and an in-house modified procedure employing the Exosomal RNA Kit from Norgen Biotek Corp. The analysis of selected gene transcripts and protein markers of extracellular vesicles (EVs) revealed that each method isolates a different mixture of uEV protein markers. In our conditions, the extraction with Norgen's reagent achieved the best performance in terms of gene transcript and protein detection and reproducibility. By using this method, we were able to detect alterations of EVs protein markers in urine samples from prostate cancer adenoma patients. Taken together, our results show that the isolation of uEVs is feasible from small volumes of urine and avoiding ultracentrifugation, making easier the analysis in a clinical facility. However, caution should be taken in the selection of the enrichment method since they have a differential affinity for protein uEVs markers and by extension for different subpopulation of EVs.
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Derio, Spain.|Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Madrid, Spain.
authors.forename: Felix
authors.initials: F
authors.lastname: Royo
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Derio, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Zuñiga-Garcia
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Derio, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Sanchez-Mosquera
authors.affiliation: Basque Biobank, Basque Foundation for Health Innovation and Research-BIOEF, Barakaldo, Spain.
authors.forename: Ainara
authors.initials: A
authors.lastname: Egia
authors.affiliation: Department of Urology, Basurto University Hospital, Bilbao, Spain.
authors.forename: Amparo
authors.initials: A
authors.lastname: Perez
authors.affiliation: Department of Urology, Basurto University Hospital, Bilbao, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Loizaga
authors.affiliation: Department of Urology, Basurto University Hospital, Bilbao, Spain.
authors.forename: Raquel
authors.initials: R
authors.lastname: Arceo
authors.affiliation: Department of Urology, Basurto University Hospital, Bilbao, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Lacasa
authors.affiliation: Department of Urology, Basurto University Hospital, Bilbao, Spain.
authors.forename: Ainara
authors.initials: A
authors.lastname: Rabade
authors.affiliation: Basque Biobank, Basque Foundation for Health Innovation and Research-BIOEF, Barakaldo, Spain.
authors.forename: Edurne
authors.initials: E
authors.lastname: Arrieta
authors.affiliation: Basque Biobank, Basque Foundation for Health Innovation and Research-BIOEF, Barakaldo, Spain.
authors.forename: Roberto
authors.initials: R
authors.lastname: Bilbao
authors.affiliation: Department of Urology, Basurto University Hospital, Bilbao, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Unda
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Derio, Spain.|Ikerbasque, Basque Foundation for Science, Bilbao, Spain.
authors.forename: Arkaitz
authors.initials: A
authors.lastname: Carracedo
authors.affiliation: CIC bioGUNE, Bizkaia Technology Park, Derio, Spain.|Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (Ciberehd), Madrid, Spain.|Ikerbasque, Basque Foundation for Science, Bilbao, Spain; jfalcon@cicbiogune.es.
authors.forename: Juan M
authors.initials: JM
authors.lastname: Falcon-Perez
keywords: exosomes; isolation of vesicles; mRNA; prostate cancer; protein markers; urine