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

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Protocol publication

[…] Quantitative RT-PCR experiments were performed using Stratagene MX 3005P thermal cycler (Agilent Technologies, Santa Clara, California, USA). Final reaction volume was 25 µl and composed of 1X SyberGreen (GoTaq® qPCR Master Mix, Promega Corporation, Madison, WI, USA or Perfecta SyberGreen FastMix®, ROX TM, Quanta BioSciences, Gaithersburg, USA), reference ROX dye, 1 µM of forward and reverse primer, and 2.16 ng of cDNA template. A first incubation was made at 50°C for 2 min followed by an initial denaturation at 95°C for another 2 min. Amplification was then performed during 40 cycles of denaturation at 95 °C for 15 sec and annealing/extension at 60 °C for 1 min. At the end, a dissociation curve was produced to analyze and confirm the specificity of the amplification by observation of a single peak in the curve. Standard curves of five points were produced for each gene to transform sample’s Cts to relative expression values. Amplification efficiencies were calculated using MxPro software (version 4.10) to analyze the slopes of the standard curves according to Pfaffl and are presented in . All samples were run in duplicate and the mean values were used for calculations. PCR products were initially sequenced to confirm the specificity of amplifications. Relative quantities of the unknown samples were normalized against the normalization factor (NF) which was calculated from the geometric means of the expression of the best three and two reference genes selected by geNorm or Normfinder algorithm respectively.GeNorm analysis determines the pairwise variation of a gene with the other tested genes, through calculation of standard deviation of the log-transformed expression ratios, and defines the internal control gene-stability measure M as the average pairwise variation of a particular gene with all other genes . The gene with the most stable expression gene has the lowest M value. A stepwise exclusion of the least stable gene allows a recalculation of the M values for the rest of genes. To determine the optimal reference gene number for normalization, we considered 0.15 as the cut-off for pairwise variation (Vn/n+1), between NFn and NFn+1 as recommended by Vandesompele et al .Normfinder is a mathematical model of gene expression that enables estimation of the overall variation of the candidate normalization genes and of the variation between sample subgroups of the sample set . Indeed, Normfinder selects the best gene for normalization based on its intra- and inter-group variation and combines the two into a stability value. It also determines the best combination of two genes which are chosen with a similar-sized fold change but in the opposite direction. [...] Ten candidate genes were tested based on their common use in literature as reference genes , , . Gene-specific, intron-spanning whenever possible, primer pairs were designed using Beacon Designer software, version 7.5 (Premier Biosoft International) or adapted from earlier publications . Primer characteristics are described in and for reference genes and genes of interest, respectively. A minimum of two designed primer pairs were tested for optimization. Primers were synthesized by Integrated DNA Technologies Inc (California, USA). Gene expression stability was tested using the geNorm algorithm which is included in the qbase Plus 2 software (version 2.3, Biogazelle, Zwijnaarde, Belgium), and Normfinder which is a free Visual Basic application (VBA) for Excel. […]

Pipeline specifications

Software tools NormFinder, Beacon Designer
Application qPCR
Organisms Oryctolagus cuniculus
Diseases Ventricular Dysfunction, Left
Chemicals Cholesterol, Ergocalciferols, NADP