Computational protocol: Validation of Suitable Reference Genes for Expression Studies in Different Pilocarpine-Induced Models of Mesial Temporal Lobe Epilepsy

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Protocol publication

[…] To assess the stability of candidate reference genes, 3 commonly used approaches geNorm (http://medgen.ugent.br/~jvdesomp/genorm/), NormFinder (http://www.mdl.dk/publicationsnormfinder.html) and Bestkeeper (http://www.wzw.tum.de/gene-quantification/bestkeeper.html) algorithms were utilized.In geNorm and NormFinder, Ct values were converted into relative quantities via the delta-Ct method using the sample with the lowest Ct as calibrator, accordling with the 2–ΔCt method . For Bestkeeper program, the raw Ct values were used.GeNorm uses an algorithm to calculate the M value, a gene expression stability factor, defined as the mean pairwise variation for a given gene compared to the remaining tested genes. Hence, a lower M value indicates higher stability of the reference gene. We considered 0.5 as a cut-off for M value because it is the smallest value that is higher than the stability values of all reference genes tested . The program also estimates the pairwise variation between two sequential calculations of normalization factors (NF) including an increasing number of genes. This defines the minimal number of genes required to calculate a robust normalization factor. NormFinder uses an ANOVA-based model to estimate intra- and inter-group variation, and combines these estimates to provide a direct measure of the variation in expression for each gene. Bestkeeper generates an index using the geometric mean of the Ct values of best candidate genes under study. This index was then compared to each individual candidate housekeeping gene by pair-wise correlation analyses, with each combination assigned a value for the Pearson correlation coefficient (r) and the probability (p). […]

Pipeline specifications

Software tools NormFinder, BestKeeper
Application qPCR
Organisms Rattus norvegicus
Diseases Epilepsy, Temporal Lobe
Chemicals Pilocarpine