Computational protocol: Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses

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Protocol publication

[…] First, three analytical tools, geNorm (version 3.5) [], NormFinder (version 0.953) [], and BestKeeper (version 1) [], were used to calculate the expression stabilities of the 15 reference genes in all samples from the different treatments.geNorm determines the most stable reference genes from a set of tested genes in a given sample set under a certain experimental condition and calculates the gene expression normalization factor via geometric averaging of all the reference genes []. geNorm evaluates gene expression stability by the M value, and ranks the tested genes according to their expression stability by stepwise exclusion of the gene with the highest M value. The lower the M value, the more stable the gene’s expression. The optimal number of reference genes was determined by pairwise variation (V) with a threshold of 0.15 []. If the value of Vn/n+1 was below 0.15, additional genes were not required for normalization. NormFinder is an algorithm based on a mathematical model of gene expression and uses a solid statistical framework to estimate the expression variation of candidate normalization genes, as well as the variation in sample subgroups, to identify the optimal normalization gene among a set of candidates []. A stability value, which is a direct measure of the estimated expression variation for each gene, is provided by NormFinder; thus, the systematic errors introduced during the process of normalization can be evaluated. In geNorm and NormFinder, the quantities obtained from Ct (cycle threshold) values via the delta-Ct method were used for data input, whereas Bestkeeper used raw Ct values. BestKeeper uses the standard deviation (SD) and coefficient of variance (CV) of the Ct values to calculate the stability of a candidate reference gene; the highest SD and CV values equate to the least stable reference genes [].Finally, RefFinder (, which is a comprehensive web-based tool that integrates geNorm, NormFinder, BestKeeper, and the comparative delta Ct method, was applied to determine the most suitable reference genes for the overall final ranking []. […]

Pipeline specifications

Software tools NormFinder, BestKeeper
Application qPCR
Chemicals Abscisic Acid, Salicylic Acid