Pre-analytical Considerations for Clinical Assays of Circulating Human miRNA-451a for Diagnostic Purposes.

Abstract

Circulating miRNA-451a has recently emerged as an important biomolecule with potential clinical values as a diagnostic marker for several diseases. However, to be used as such, it is critical to accurately assay miRNA-451a in the clinic. Yet, pre-analytical factors that can affect such an error-free clinical assay of miRNA-451a have not been explored. This study aimed at investigating several of these pre-analytical factors that may affect the accurate quantification of miRNA-451a in human blood samples. We initially evaluated miRNA-451a levels in red blood cells (RBCs), white blood cells (WBCs), platelets, and plasma by droplet digital PCR (ddPCR). Next, we quantified miRNA-451a levels in whole blood or plasma stored at different temperatures for different time periods by ddPCR and used hemolyzed plasma samples to study the effects of hemolysis on plasma miRNA-451a concentration. We found that WBCs and platelets contain trace amounts of miRNA-451a and that about 99.9% of miRNA-451a in the blood are localized in RBCs. Hemolyzed plasma showed 58.5-fold increase in miRNA-451 concentration as compared to non-hemolyzed plasma. Importantly, plasma stored at room temperature (RT) or 4ºC showed a steady statistically significant decrease in miRNA-451a levels over a 7-day period whereas plasma samples stored at -20 or -80ºC for up to 7 days showed stable miRNA-451a levels. Together, our data indicate that hemolysis and blood storage at RT or 4ºC may have significant negative effects on the levels of circulating miRNA-451a.