Urinary extracellular vesicles isolated by ultrafiltration combined with size exclusion chromatography for small RNA sequencing analysis

Urinary extracellular vesicles (EVs) are potential biomarkers for the early diagnosis of urinary system diseases, but their clinical translation still requires optimization of the EVs isolation process. Size exclusion chromatographic (SEC) is a well-established method for the isolation of EVs, which can obtain high-purity EVs from plasma. In this work, home-made SEC columns were used for the isolation of urinary EVs, and the optimized SEC column can achieve high-efficiency separation of urinary EVs from impurity proteins. Compared with ultracentrifugation (UC) method, SEC can achieve higher EVs recovery rate and purity. RNA-sequencing was performed to assess the value of SEC in clinical biomarker screening, and the results showed that the isolation method has little effect on the sequencing results of EVs. However, the SEC method offered a shorter process time, less operation requirements and better batch-to-batch stability of isolated EVs than UC, thus having a higher potential in clinical translational applications.