Normalization strategy for selection of reference genes for RT-qPCR analysis in left ventricles of failing human hearts

Abstract Background Quantitative RT-PCR is a valuable tool for assessing the gene expression in different human tissues, particularly due to its exceptional sensitivity, accuracy and reliability. However, the choice of adequate control for normalization is a crucial step, greatly affecting the results of all subsequent analyses. So far, only a few studies were focused on the selection of optimal reference genes in left ventricles of failing human hearts, leading to several disparities in experimental results focused on differential gene expression in this area. Therefore, the main objective of this study was to identify a set of suitable reference genes in normal and failing left ventricle tissues, which could increase the reliability of RT-qPCR-based studies in the future. Methods We analyzed the expression of 15 commonly used housekeeping genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, IPO8, PGK1, POLR2A, PPIA, RPLP0, TBP, TFRC, UBC and YWHAZ) in left ventricles of normal and failed hearts with two-step approach. In the first step, we excluded genes which are variantly expressed using ANOVA-based statistical method. Afterwards, the remaining genes were analyzed using geNorm, NormFinder and BestKeeper algorithms, together with delta Cq method. Finally, the geometric mean of gene rankings across all methods was calculated. Results Our analysis identified IPO8 and POLR2A as the most stably expressed genes, whereas ACTB and B2M were found to be expressed variantly, suggesting a potential role of these genes in the pathophysiological processes in failing human hearts. Discussion/conclusion Using our two-step approach, we identified and validated two reference genes expressed invariantly in left ventricles of both healthy and failing human hearts, as well as provided a guideline for the selection of reference genes in studies comparing gene expression in these types of tissues.