Abstract
Extracellular vesicle (EV) miRNAs are promising biomarkers for clinical diagnosis. However, their stability is a crucial concern affecting reliability and accuracy. Factors such as sample collection, processing, storage conditions, and experimental procedures impact EV miRNA stability. Studying EV miRNA stability aims to find optimal handling and storage methods, ensuring integrity and functionality throughout research. In this study, we used RT-qPCR and GlyExo-Capture technology, which can specifically capture glycosylated EVs by lectin, to assess the stability of glycosylated EV miRNAs. We found that slow acceleration centrifugation and two-step centrifugation methods were suitable for subsequent experiments. To ensure uniformity, we recommend using the two-step centrifugation method. We also studied blood storage before serum separation and recommend separation within 2 h at 4 °C or 25 °C. For separated serum samples, higher temperatures accelerated miRNA degradation, and the storage duration should be adjusted based on laboratory conditions. Short-term storage at -20 °C is acceptable for up to 3 months while avoiding repeated freeze-thaw cycles. We developed protective agents to extend the storage time at 25 °C, meeting clinical requirements. Additionally, Lakebio's cfRNA storage tubes effectively preserved the stability of miRNAs in plasma glycosylated EVs. Understanding EV miRNA stability provides insights into optimizing sample handling, storage strategies, and enhancing reliability in clinical applications.