Computational protocol: Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don

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Protocol publication

[…] Standard curves were used to determine the gene-specific PCR efficiency level from a 10-fold dilution of the mixed cDNA template for each primer pair. Correlation coefficients (R2) and slope values were obtained from the standard curve, and corresponding PCR amplification efficiencies (E) were determined according to the following equation: E = (10-1/slope-1) × 100 ().Reference gene expression stability was statistically analyzed based on the results obtained from the following three Excel-based software programs: GeNorm (), NormFinder and BestKeeper (; ). The GeNorm program was used to calculate the expression stability value (M) for reference genes based on the pairwise variation. Stable expression genes were characterized with lower M values. The BestKeeper program uses an index for the evaluation of expression stability that is calculated based on standard deviation (SD) and percentage covariance (CV) values (). The NormFinder program can determine degrees of variance within and between groups, and the gene with the lowest stability value is ranked as the best. […]

Pipeline specifications

Software tools NormFinder, BestKeeper
Application qPCR