Computational protocol: Evaluation of Reference Genes for RT-qPCR Studies in the Seagrass Zostera muelleri Exposed to Light Limitation

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[…] Data from RT-qPCR was analysed using the Step One Plus Software (Ver. 2.3; Applied Biosystems). Expression levels were determined as the number of cycles needed for the amplification to reach a fixed threshold in the exponential phase of the RT-qPCR reaction. The cycle threshold (CT) was set at 0.03 for all genes. The corresponding CT values were used directly in the software packages geNorm, NormFinder and Bestkeeper to rank and select the most stable reference genes. To validate changes in target genes expression, CT were imported into the qbase+ software package (Biogazelle) and transformed into quantities using maximum efficiency of 1.00 (or 100%) to obtain Calibrated Normalized Relative Quantities (CNRQ). [...] We established a consensus rank of genes by combining stability measurements produced by geNorm, NormFinder and BestKeeper by using the RankAggreg package (http://cran.rproject.org/web/packages/RankAggreg/index.html). Briefly, we imported the matrix of rank-ordered genes according to the 3 different software packages to the RankAggreg statistical package, calculated the “distance” between ordered lists using the Spearman foot rule function and performed rank aggregation via the Cross-Entropy Monte Carlo algorithm. The consensus ranking with the lowest score was then chosen. [...] Statistical analyses were done using the software Statistica 7.0 (Statsoft Inc., Tulsa, OK, USA). Kolmogorov-Smirnov and Levene’ test were used to first assess the data for normality and homoscedasticity, respectively. The data was found to be normal and homogenous, and therefore parametric test (t-test) was used to test the effect of light limitation on the expression level of target genes. Throughout this paper, values given are the mean of 3 biological replicates (each biological replicate being a sample pool of 2–4 plants each), including technical triplicates for RT-qPCR data. Results were considered significant at 5%. […]

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