Computational protocol: Analysis of Different Approaches for the Selection of Reference Genes in RT-qPCR Experiments: A Case Study in Skeletal Muscle of Growing Mice

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Protocol publication

[…] Primers were designed with the available free software Primer BLAST [] and OligoPerfectTM Designer (Thermo Fisher Scientific, Inc.) following general recommendations: 55–65 °C melting temperature, 18–25 base pair length, 45–60% GC content and delimiting a 50–150 base pairs length amplicon. Primers that fulfilled these criteria were chosen, with the exception of GAPDH forward primer that contains 67% of GC and ACTB primer pair that amplifies a 319 base pairs length sequence ( and ). To avoid amplification of potentially remaining genomic DNA, primers that span exon–exon junctions were chosen whenever possible. Moreover, no-reverse transcription controls were always performed and showed no amplification product.Primer and amplicon secondary structures were evaluated using OligoAnalyzer 3.1 Software (Integrated DNA Technologies, Inc., Coralville, IA, USA) in order to select primers that do not form hairpins, self-dimers or heterodimers and that do not anneal to amplicon regions involved in hairpins. In addition, primers that amplified regions that could form stable secondary structures were rejected. Specificity of primers was checked by carrying out a BLAST search with Primer BLAST software. Primers were obtained from Integrated DNA Technologies, Inc. and InvitrogenTM. [...] Quantification cycle (Cq) determination was performed with the Applied Biosystems® 7500 Software v2.0.6 with automatic baseline correction and threshold setting. Prior to analyzing Cq data, the automatic threshold setting was evaluated and, if needed, adjusted according to general recommendations. Relative gene expression levels were calculated by the comparative Cq method [], which refers results from experimental samples to a calibrator according to the equation EA−B (E = amplification efficiency = 10(−1/slope), A = average Cq value of the corresponding run, B = mean Cq value of the sample). Data were then expressed as fold change related to the mean of 9-week-old normal mice and analyzed by the D’Agostino-Pearson normality test to assess for Gaussian distribution. For those data sets that did not pass this test, logarithmic transformation was performed to achieve normality. The significance of the difference in the mean level for each transcript among ages and genotypes was assessed by two-way analysis of variance (ANOVA) or by one-way ANOVA when only normal mice were analyzed, followed by the Bonferroni post-test where a p-value lower than 0.05 was considered statistically significant. Differences among ages are expressed by different letters, small letters for normal animals and capital letters for transgenic mice. Differences between genotypes are expressed by an asterisk. This statistical analysis was performed using the GraphPad PrismTM 5.01 program (GraphPad Software, Inc., La Jolla, CA, USA).In order to determine the most stable genes among the set of potential reference genes, data were also analyzed with different statistical algorithms available as visual basic applications for Microsoft Excel: geNorm (version 3) [], NormFinder (version 0953) [], BestKeeper (version 1) [] and the Comparative ΔCq method []. These methods yield a ranking based on stability values for each gene. […]

Pipeline specifications

Software tools Primer-BLAST, OligoAnalyzer, NormFinder, BestKeeper
Application qPCR
Organisms Mus musculus