Pre-Analytical Handling Conditions and Small RNA Recovery from Urine for miRNA Profiling

There are currently no standardized protocols for pre-analytical handling of urine to best preserve small RNA for miRNA profiling studies. miRNA is an attractive candidate as a potential biomarker because of the high level of stability in body fluids and its ability to be quantified on multiple high-throughput platforms. We present a comparison of small RNA recovery and stability in urine under alternate pre-analytical handling conditions and extend recommendations on what conditions optimize yield of miRNA from cell-free urine and urine extracellular vesicles (EVs). Using an affinity slurry for isolation of small RNA from urine, we found that urine samples held at room temperature (20 degrees C) for up to 8 hours before processing yield the highest amounts of intact small RNAs from EVs. Some miRNA is lost from urine samples when held 2 degrees C to 4 degrees C and/or frozen before EV isolation, likely because of EV entrapment in uromodulin precipitates. However, we found that a simple 5-minute incubation of urine containing cold-induced precipitate at 37 degrees C resolubilizes much of this precipitate and results in an increased recovery of EVs and miRNAs. Finally, small RNA integrity can be compromised when whole urine is held at 37 degrees C for as little as 4 hours and is not conducive to efficient miRNA profiling.