Stability of microRNAs in serum and plasma reveal promise as a circulating biomarker.

Purpose: To verify the stability and reliability of circulating microRNA (miRNA) profiles in plasma and serum under different processing and storage conditions to inform future applications to circulating biomarker analyses. Background: The development of blood-based methods for early disease detection has become increasingly desirable across various medical fields. RNA profiles have been investigated but have been a challenge due to rapid degradation of the analyte by ubiquitous RNases. miRNAs are short, non-coding regulatory RNAs that are believed to be more stable under certain conditions, large in number, and specific to cell type and disease state. Thus, circulating miRNA profiles hold significant promise as diagnostic biomarkers for a range of conditions, including cancer, autoimmune, liver, neurological, metabolic, and cardiovascular diseases. However, to realize their full potential in clinical applications, it is crucial to thoroughly characterize the stability of miRNAs under various blood collection, processing, and storage conditions prior to their investigation and large-scale application in disease-specific biomarker discovery studies. Methods: Plasma or serum were extracted from whole blood of healthy volunteers. Samples were stored at different temperatures (4 °C or 25 °C, room temperature) for varying periods (0-24 h) to mimic possible delays in processing encountered in routine clinical settings. miRNA was extracted and profiles were assessed with RT-qPCR or small RNA-sequencing techniques. Results: Mean Cq values of specific miRNAs, such as miR-15b, miR-16, miR-21, miR-24, and miR-223, remained consistent between 0 and 24 h when serum and plasma were stored on ice. Minimal changes were observed in mean Cq values over 24 h when serum was left at room temperature as well. Similar trends were observed when miRNAs from plasma were analyzed. Small-RNA sequencing detected approximately ∼650 different miRNA signals in plasma, with over 99 % of the miRNA profile unchanged even when blood draw tubes were left at room temperature for 6 h prior to processing. Conclusions: These data demonstrate remarkable stability of miRNAs over time, which should withstand variability in handling and processing that can occur with routine clinical lab draws. Considering the large diversity of miRNAs, this analyte class should be thoroughly investigated as a non-invasive biomarker of diverse disease states.