Identification of Appropriate Endogenous Controls for Circulating miRNA Quantification in Working Dogs under Physiological Stress Conditions

SIMPLE SUMMARY: Circulating miRNAs are not only present in cells but also in the extracellular environment, especially in different biofluids including blood, and can act in a paracrine manner by facilitating a diversity of signaling mechanisms between cells. In qPCR gene expression profiling analysis, the endogenous control must be a stable gene to allow an accurate cross-sample gene expression comparison. The appropriate selection of an endogenous control is a crucial step. This research aims to select, in the miRNome, appropriate circulating miRNAs that can serve as an endogenous control. The study model are working dogs used in the search and rescue of missing persons after natural disasters. ABSTRACT: Cell-free miRNAs, called circulating miRNAs (cmiRNAs), can act in a paracrine manner by facilitating a diversity of signaling mechanisms between cells. Real-time qPCR is the most accepted method for quantifying miRNA expression levels. The use of stable miRNA endogenous control (EC) for qPCR data normalization allows an accurate cross-sample gene expression comparison. The appropriate selection of EC is a crucial step because qPCR data can change drastically when normalization is performed using an unstable versus a stable EC. To find EC cmiRNA with stable expression in search and rescue (SAR) working dogs, we explored the serum miRNome by Next-Generation Sequencing (NGS) at T0 (resting state) and T1 immediately after SAR performance (state of physiologically recovered stress). The cmiRNAs selected in the NGS circulating miRNome as probable ECs were validated by qPCR, and miRNA stability was evaluated using the Delta Ct, BestKeeper, NormFinder, and GeNorm algorithms. Finally, RefFinder was used to rank the stability orders at both T0 and T1 by establishing miR-320 and miR-191 as the best-circulating ECs. We are confident that this study not only provides a helpful result in itself but also an experimental design for selecting the best endogenous controls to normalize gene expression for genes beyond circulating miRNAs.