Different EV enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles

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.

Scientists at CIC bioGUNE 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 their 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, they were able to detect alterations of EVs protein markers in urine samples from prostate cancer adenoma patients. Taken together, these 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.

Experimental design for comparing 5 uEVs enrichment methods

Two 10-ml aliquots of urine from 10 healthy individuals were used to test each of the 5 methods (overall, 100 samples were processed). The final pellet obtained in each case was suspended in 100 µl of Exosome Resuspension Buffer (ERB, Life Technologies) and then divided for RNA, protein and EM analysis at the ratio of 70:20:10. The same ratio was used for NOR extraction, before proceeding to the first centrifugation.