Identification of suitable controls for miRNA quantification in T-cells and whole blood cells in sepsis.

ABSTRACT

Complex immune dysregulation is a hallmark of sepsis. The occurring phases of immunosuppression and hyperinflammation require rapid detection and close monitoring. Reliable tools to monitor patient's immune status are yet missing. Currently, microRNAs are being discussed as promising new biomarkers in sepsis. However, no suitable internal control for normalization of miRNA expression by qPCR has been validated so far, thus hampering their potential benefit. We here present the first evaluation of endogenous controls for miRNA analysis in human sepsis. Novel candidate reference miRNAs were identified via miRNA microArray. TaqMan qPCR assays were performed to evaluate these microRNAs in T-cells and whole blood cells of sepsis patients and healthy controls in two independent cohorts. In T-cells, U48 and miR-320 proved suitable as endogenous controls, while in whole blood cells, U44 and miR-942 provided best stability values for normalization of miRNA quantification. Commonly used snRNA U6 exhibited worst stability in all sample groups. The identified internal controls have been prospectively validated in independent cohorts. The critical importance of housekeeping gene selection is emphasized by exemplary quantification of imuno-miR-150 in sepsis patients. Use of appropriate internal controls could facilitate research on miRNA-based biomarker-use and might even improve treatment strategies in the future.