Computational protocol: Selection of Reliable Reference Genes in Caenorhabditis elegans for Analysis of Nanotoxicity

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Protocol publication

[…] For each primer set, standard curves made from serial dilutions of pooled cDNA were used to estimate PCR reaction efficiency (E) using the formula: E (%) = (10[−1/slope]−1)×100. The expression stability of the 13 candidate genes were evaluated using four commonly used algorithms, geNorm , NormFinder , BestKeeper , and the comparative ΔCt method . The overall ranking of candidate reference genes was generated according to a method reported previously .GeNorm (version 3.5) is an Excel-based applet that can be used for the analysis of gene expression stability and eventually providing two most stable reference genes. The values of transformed Ct (relative expression values) were transferred into the geNorm applet as input data. The expression stability value (M value) was calculated by the geNorm program for each candidate gene, which is described as the average pairwise variation of a single candidate reference gene to all other tested genes. A low M value indicates high stability in gene expression, thereby maybe ideal reference genes. Furthermore, geNorm can also provide the minimal number of reference genes required for reliable normalization. According to the pairwise variation calculation, 0.15 is commonly accepted as the cutoff, below which an additional reference gene is not required for accuracy normalization .Another Excel-based software NormFinder was also used to identify reference genes for optimal normalization. This approach has the advantage of ranking the candidate reference genes both inter-group and intra-group according to their different expression stability.Also an Excel-based software BestKeeper was used to evaluate the expression stability of candidate reference genes. This program creates an index using the geometric mean of each candidate gene's raw Ct values. Gene expression variation can be determined by the calculated standard deviation (SD) and coefficient of variance (CV) for all candidate reference genes based on their Ct values. Candidate genes with SD values greater than 1 were considered as inconsistent and were excluded. Then the BestKeeper program estimated the relationship between the index and the contributing reference gene by the Pearson correlation coefficient, the coefficient of determination (r2), and the P value.Moreover, the comparative ΔCt method was used to estimate the most stable reference genes. By comparing the relative expression of “pairs of genes” within each treatment, this method indicated the mean of standard deviation of each candidate reference genes. The candidate with lowest SD value was proposed to be the most stable gene and the highest SD value indicated the least stable gene. […]

Pipeline specifications

Software tools NormFinder, BestKeeper
Application qPCR
Organisms Caenorhabditis elegans