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.

• urine
• exosomes
• isolation of vesicles
• mRNA
• protein markers
• prostate cancer

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Acknowledgements

We thank David Gil of the CIC bioGUNE electron microscopy platform for his technical assistance in the cryo-EM analysis. We are thankful to the Basque Biobank for Research (BIOEF) funded by ISCIII (PT13/0010/0052) for the acquisition, maintenance and distribution of urine samples, and to the patients enrolled in this study. The work was supported by ISCIII (PI12/01604), ISCIII (PI10/01484, PI13/00031), GV Health (2012111086) and Education (PI2012-03), Marie Curie (277043), ERC (336343) and the Movember Foundation (GAP1). Centro de Investigación Biomédica en Red en el Área temática de Enfermedades Hepáticas y Digestivas (CIBERehd) is funded by the Spanish ISCIII-MICINN.

Notes

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