Circulating miRNAs are detected in extracellular space and body fluids such as urine. Circulating RNAs can be packaged in secreted urinary extracellular vesicles (uEVs) and thus protected from degradation. Urinary exosome preparations might contain specific miRNAs, relevant as biomarkers in renal and bladder diseases. Major difficulties in application of uEVs into the clinical environment are the high variability and low reproducibility of uEV isolation methods.
University of Bern researchers used five different methods to isolate uEVs and compared the size distribution, morphology, yield, presence of exosomal protein markers and RNA content of uEVs. They present an optimized ultracentrifugation and size exclusion chromatography approach for highly reproducible isolation for 50-150 nm uEVs, corresponding to the exosomes, from 50 ml urine. They profiled the miRNA content of uEVs and total urine from the same samples with the NanoString platform and validated the data using qPCR. These results indicate that 18 miRNAs, robustly detected in uEVs were always present in the total urine. However, 15 miRNAs could be detected only in the total urine preparations and might represent naked circulating miRNA species. This is a novel unbiased and reproducible strategy for uEVs isolation, content normalization and miRNA cargo analysis, suitable for biomarker discovery studies.
The optimized UC-SEC method overview
Midstream, clean catch first-morning urine was collected. Urine samples (50 ml) were mixed with 4.2 ml of protease inhibitor (PI) solution immediately after urine collection. Urine was vortexed and centrifuged at 200 × g for 20 min to remove cells. After discarding the pellet, the cell-free urine was centrifuged at 2000 × g for 20 min to remove cell debris and large protein aggregates. Supernatant was centrifuged at 16000 × g for 20 min to remove ectosomes and other large particles. Supernatant was collected and kept in 4 °C. The pellet was treated with DTT (200 mg/ml) and incubated 10 min in 37 °C. After a short vortex the mixture was centrifuged at 16000 × g for 20 min and supernatant was pooled with the supernatant from last 16000 × g centrifugation step. The pooled supernatants were filtered through 0.22 μm filters and ultracentrifuged at 120000 × g for 70 min. The supernatant was carefully decanted, the pellet overlaid with particle-free PBS and subjected to ultracentrifugation at 120000 × g for 70 min. The final pellet was resuspended in 500 μl of particle-free PBS. The resuspended pellet was used for SEC, NTA, protein quantification and morphology evaluation by EM.