Systematic evaluation of microRNA stability during long term storage of urine

Despite their promising utility as biomarkers for disease prediction, the stability of microRNAs (miRNAs) encapsulated in urinary extracellular vesicles (uEVs) during long-term storage remains insufficiently characterized, limiting both research throughput and clinical application. Here, we systematically evaluated the stability of urine stored for up to 12 months, as well as the stability of uEVs, uEV-derived miRNAs, and cDNA libraries for up to 3 months. Next generation sequencing-based profiling demonstrated that the abundance of miRNAs and participant-specific expression profiles were largely preserved under all storage conditions during the observation periods. At the individual miRNA level, expression patterns were generally maintained for 12 months in urine and for 3 months in uEVs and cDNA libraries, whereas partial degradation was observed in samples stored as RNA. A single freeze–thaw cycle did not significantly affect the miRNA yield or profiles in urine, uEVs, miRNAs, or cDNA libraries. These findings demonstrate that urine samples can be stored at − 80 °C for at least 12 months without major profile shifts, supporting the broad feasibility of uEV-derived miRNAs in research and clinical applications.