title: 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.
authors.affiliation: Montreal Heart Institute and Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Walid
authors.initials: W
authors.lastname: Nachar
authors.affiliation: Montreal Heart Institute and Université de Montréal, Montreal, Quebec, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Busseuil
authors.affiliation: Montreal Heart Institute and Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Yanfen
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Montreal Heart Institute and Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Teodora
authors.initials: T
authors.lastname: Mihalache-Avram
authors.affiliation: Montreal Heart Institute and Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Mecteau
authors.affiliation: Montreal Heart Institute and Université de Montréal, Montreal, Quebec, Canada ; Department of Medicine, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Eric
authors.initials: E
authors.lastname: Rhéaume
authors.affiliation: Montreal Heart Institute and Université de Montréal, Montreal, Quebec, Canada ; Department of Medicine, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Tardif
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis; D059305:Diet, High-Fat; D004195:Disease Models, Animal; D004452:Echocardiography; D020869:Gene Expression Profiling; D056726:Genetic Association Studies; D005819:Genetic Markers / Q000235:genetics*; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction; D018487:Ventricular Dysfunction, Left / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D005819:Genetic Markers