Optimisation of Reference Genes for Gene-Expression Analysis in a Rabbit Model of Left Ventricular Diastolic Dysfunction

Abstract

Left ventricular diastolic dysfunction (LVDD) is characterized by the disturbance of ventricle’s performance due to its abnormal relaxation or to its increased stiffness during the diastolic phase. The molecular mechanisms underlying LVDD remain unknown. We aimed to identify normalization genes for accurate gene-expression analysis of LVDD using quantitative real-time PCR (RT-PCR) in a new rabbit model of LVDD. Eighteen rabbits were fed with a normal diet (n = 7) or a 0.5% cholesterol-enriched diet supplemented with vitamin D2 (n = 11) for an average of 14.5 weeks. We validated the presence of LVDD in this model using echocardiography for diastolic function assessment. RT-PCR was performed using cDNA derived from left ventricle samples to measure the stability of 10 genes as candidate reference genes (Gapdh, Hprt1, Ppia, Sdha, Rpl5, Actb, Eef1e1, Ywhaz, Pgk1, and G6pd). Using geNorm analysis, we report that Sdha, Gapdh and Hprt1 genes had the highest stability (M <0.2). By contrast, Hprt1 and Rpl5 genes were found to represent the best combination for normalization when using the Normfinder algorithm (stability value of 0.042). Comparison of both normalization strategies highlighted an increase of natriuretic peptides (Bnp and Anp), monocytes chemotactic protein-1 (Mcp-1) and NADPH oxidase subunit (Nox-2) mRNA expressions in ventricle samples of the hypercholesterolemic rabbits compared to controls (P<0.05). This increase correlates with LVDD echocardiographic parameters and most importantly it molecularly validates the presence of the disease in our model. This is the first study emphasizing the selection of stable reference genes for RT-PCR normalization in a rabbit model of LVDD.

Selection of reference genes is critical for miRNA expression analysis in human cardiac tissue. A focus on atrial fibrillation

“…A large number of studies have been carried out concerning the validation of reference genes in different tissue and cell types192021222324. Nonetheless, studies on the best normalization strategy in cardiac tissue remain sparse252627, and, to the best of our knowledge, no previous study has been performed to identify appropriate reference genes in the human atrial tissue for the study of AF. The lack of a general consensus on the best normalization strategy in this context has significant impact, considering the large increase in the number of studies analyzing the relationship between miRNA and AF in the last five years789.…”

Importance of reference gene selection for articular cartilage mechanobiology studies

“…A real-time qPCR assay based on SYBR green detection, using Brilliant III Ultra-Fast SYBR ® QPCR mix (Agilent Technologies, Stockport, UK) was used for the transcriptional profiling of eight reference genes including 18s 16 , GAPDH 17 , ACTB, HPRT, SDHA (Succinate dehydrogenase complex, subunit A), RPL4 (Ribosomal Protein L4), PPIA and YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein) 18 . The choice of selected reference gene targets for analysis was largely based on reference gene suitability previously analysed in loaded, isolated chondrocytes 3 , reported to be stable under mechanical perturbation in other tissues 19 , 20 or reported to be stable in chondrocytes under other experimental conditions 21 . In addition, the analysis of two commonly examined cartilage genes matrix metalloproteinase 3 (MMP3) 22 and aggrecan (ACAN) 17 ( Table I ) were performed.…”

The Impact of a Non-Functional Thyroid Receptor Beta upon Triiodotironine-Induced Cardiac Hypertrophy in Mice

“…Hyperthyroidism was induced by 3 weeks of daily T3 (3,5,3´-Triiodo-L-Thyronine; SigmaAldrich, SP, Brazil) subcutaneous (s.c.) injections, at increasing doses of 0.2, 0.5 and 1.0 µg/100 g body weight (bw), after the initial 5 weeks of hypothyroidism induction (Hyper). Hypothyroidism and hyperthyroidism protocols were followed in accordance to recent guidelines published by a group of thyroid experts [32,33]. Twenty-four hours after the final injection of vehicle or T3, mice were sacrificed by decapitation, and their sera and hearts were collected and stored at -70°C until evaluation.…”

“…Intron spanning primers were synthesized by Integrated DNA Technologies (IA, USA) and were obtained from references as shown in Table 1. 36B4 (Rplp0) was used as the endogenous control as previously described by us and validated in the heart by others [33][34][35]. Samples were analysed in duplicate, and the cycle parameters were as follows: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 45 s. The product purity was confirmed by agarose gel and melting curve analyses, and the efficiency of each assay was confirmed and accepted when near 100%.…”