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

Publication date: Available online 22 June 2018Source: The Journal of Molecular DiagnosticsAuthor(s): David A. Armstrong, John A. Dessaint, Carol S. Ringelberg, Haley F. Hazlett, Louisa Howard, Moemen A.K. Abdalla, Roxanna L. Barnaby, Bruce A. Stanton, Mark A. Cervinski, Alix AshareThere are currently no standardized protocols for pre-analytical handling of urine to best preserve small RNA for microRNA profiling studies. MicroRNA is an attractive candidate as a potential biomarker due to high level of stability in body fluids and its ability to be quantified on multiple high-throughput platforms. Here, we present a comparison of small RNA recovery/stability in urine under alternate pre-analytical handling conditions and extend recommendations on what conditions optimize yield of microRNA from cell-free urine and urine extracellular vesicles (EVs). Using an affinity slurry for isolation of small RNA from urine, we find that urine samples held at room temperature (20°C) for up to eight hours prior to processing yield the highest amounts of intact small RNAs from EVs. Some microRNA is lost from urine samples when held 2 to 4°C and/or frozen prior to EV isolation, likely due to EV entrapment in uromodulin precipitates. However, we find that a simple 5-minute incubation of urine containing ‘cold-induced’ precipitate at 37°C, resolubilizes much of this precipitate and results in an increased recovery of EVs and microRNAs. Finally, small RNA integrity can be compromised when ...

Source: The Journal of Molecular Diagnostics - July 5, 2018 Category: Pathology Source Type: research