Abstract 1476: Stability of circulating microRNA for plasma and serum biomarker studies
Authors: Katayama Erryk S. , Hue Jonathan J. , Zarei Mehrdad , Graor Hallie J. , Hajihassani Omid , Vaziri-Gohar Ali , Winter Jordan M. 
Published: June 2022

Abstract
Purpose: To verify the stability and reliability of circulating microRNA (miRNA) profiles in plasma and serum under different processing and storage conditions for the optimization of future biomarker analyses.
Background: Development of blood-based early cancer detection methods have become increasingly popular in recent years. RNA profiles have been investigated; however, obtaining reliable RNA data has been a challenge due to rapid degradation by RNases. miRNAs are short non-coding regulatory RNAs that are more stable as they are complexed with proteins or packaged in exosomes. Thus, circulating miRNA profiles have the potential to serve as a diagnostic test for cancer. Optimization of collection protocols is a crucial next step for widespread use.
Methods: Whole blood was drawn after Institutional Review Board approval. Blood was processed into either plasma or serum aliquots. The samples were stored at different temperatures (0 or 25°C) for varying periods of time (0-24 hours). miRNA was extracted using a Qiagen miRNeasy Serum/Plasma kit. Changes in profiles were assessed with RT-qPCR using a previously established panel of consistently expressed miRNAs (miR15b, miR16, miR21, miR24, miR223). Values were compared using two-tailed t-tests and values are presented as mean ± standard deviation.
Results: Mean Cq values were similar at 0 and 24 hours when serum was stored on ice, suggesting negligible miRNA degradation: miR15b (29.6±0.5 vs 29.8±0.4, p=0.11), miR16 (23.5±0.8 vs 23.3±1.0, p=0.55), miR21 (28.6±0.5 vs 28.9±0.6, p=0.17), miR24 (27.4±0.7 vs 27.0±0.5, p=0.09), and miR223 (22.9±0.5 vs 22.9±0.7, p=0.90). There was similar stability over time for 60% of tested miRNAs when serum was left at room temperature: miR15b (30.3±0.7 vs 30.7±0.9, p=0.22), miR16 (23.9±0.7 vs 24.3±1.0, p=0.28), and miR21 (30.0±0.6 vs 29.9±0.6, p=0.38). Two of the miRNAs demonstrated a statistically significant increase in mean Cq after 24 hours: miR24 (27.6±0.7 vs 28.2±0.7, p=0.03) and miR223 (23.4±0.4 vs 24.6±0.6, p=&lt;0.01). In general, these trends were similar when plasma was collected, as compared to serum, at both room temperature and on ice. miRNA profiles collected from serum were overall like those obtained from plasma.
Conclusions: These data confirm and expand upon past reports demonstrating the remarkable stability of miRNA. Even in suboptimal processing conditions (i.e., room temperature for 24 hours), the miRNA profile is remarkably consistent. Although blood processing and transport times may vary between institutions, our data suggest these discrepancies may not affect the efficacy of miRNA as a blood biomarker. We plan to validate our findings using bulk small RNA sequencing, which we hypothesize will similarly demonstrate minimal changes in the entire miRNA landscape in different conditions. We will then utilize our institutional biorepository to identify potentially unique miRNA profiles present in patients with cancer.
Citation Format: Erryk S. Katayama, Jonathan J. Hue, Mehrdad Zarei, Hallie J. Graor, Omid Hajihassani, Ali Vaziri-Gohar, Jordan M. Winter. Stability of circulating microRNA for plasma and serum biomarker studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1476.