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.
Highlights
Complex immune dysregulation is a hallmark of sepsis
An optimal housekeeping gene should fulfil several essential demands: (1) it is highly expressed in the target cell, (2) it shows no differential regulation exhibiting high expression stability over time, (3) it is unaffected by any experimental condition and (4) it is detectable by use of available assays[24,25]
Optimal internal controls according to these requirements have to be determined for each utilized type of cell or tissue and every experimental approach[20,26,27]
Summary
Complex immune dysregulation is a hallmark of sepsis. The occurring phases of immunosuppression and hyperinflammation require rapid detection and close monitoring. Various biomarkers have been proposed[6], effective tools for monitoring patients’ immune status are still missing, hampering targeted therapy of sepsis. MicroRNAs are remarkably stable and measurable[7,11] They are tissue- and cell-type-dependently expressed and specific changes in miRNA expression due to cellular damage create disease-specific miRNA signatures[4,12,13]. Accession number NR_002752 NR_002750 AF141346 NR_002745 MI0000542 MI0005767 MI0000736 www.nature.com/scientificreports were heterogeneous currently impeding the use of miRNA as biomarkers This lack of consensus might -at least partially- be due to the fact that valid reference genes for quantification of miRNAs in sepsis have not been established, yet[17,18,19,20,21,22,23]. In many cases either plasma or serum samples were used, which bears the risk of detecting contaminating miRNAs induced or released by co-morbidities or environmental influences
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