As the remodeling progresses, heart changes its shape and morphology, mainly due to pathological processes such as cardiomyocyte hypertrophy, apoptosis, inflammation, and collagen deposition.
In numerous types of heart diseases, including ischemic heart disease, dilated cardiomyopathy and coronary artery disease, ventricular remodeling is a major feature of heart failure. Heart failure (HF) is a serious clinical disorder and the most common reason for hospitalization and death among older adults in Europe and the United States. 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. 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. Finally, the geometric mean of gene rankings across all methods was calculated.
Afterwards, the remaining genes were analyzed using geNorm, NormFinder and BestKeeper algorithms, together with delta Cq method. In the first step, we excluded genes which are variantly expressed using ANOVA-based statistical method. 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. 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. 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. However, the choice of adequate control for normalization is a crucial step, greatly affecting the results of all subsequent analyses. 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.
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