Computational protocol: Validation of Reference Genes for the Determination of Platelet Transcript Level in Healthy Individuals and in Patients with the History of Myocardial Infarction

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Protocol publication

[…] During the assessment of a set of RGs, the implemented evaluation method can be a source of bias related to the assumptions underlying each approach. In an effort to minimize bias, we tested the expression stability of nine selected internal control candidate genes by four different approaches found in the literature [,–] by using RefFinder program [].ΔCq approach introduced by Silver et al. compares the relative expression of all pairwise combination of genes within each sample []. This comparison provides information on which pairs show least variability and hence which gene(s) has the most stable expression. The BestKeeper software calculates reference gene’s standard deviation (SD) based on raw Cq values regardless of sample’s efficiency []. NormFinder analysis enables estimation of the overall variation of the candidate normalization genes []. The combined measure of intra- and intergroup-variation is given as a stability value, which is an estimation of the variation in the expression of candidate RGs. The basic assumption is that a stable RG should have minimal variation across experimental groups and subgroups. Finally, geNorm is a Visual Basic Application for Microsoft Excel, which uses an algorithm to calculate M-value, a transcript level stability measure, defined as the mean pairwise variation for a given gene compared to the remaining tested genes. Stepwise exclusion of the reference gene with the least stable expression finally assigns the two most stable genes []. RefFinder is a web-based tool to select the most stably expressed mRNAs among a panel of RGs. It integrates geNorm, Normfinder, BestKeeper, and the comparative ΔCt method to rank the candidate RGs. It calculates the RG ranking based on each program, then evaluates final ranking by assigning a suitable weight to an individual gene and calculates the geometric mean of their weights. […]

Pipeline specifications

Software tools BestKeeper, NormFinder
Application qPCR
Organisms Homo sapiens
Diseases Myocardial Infarction
Chemicals Alprostadil, Glucose