Analysis of reference gene stability

Data analysis was performed with the combined set of samples (control and endometriosis groups). The stability of the candidate reference genes was estimated using the mean Ct values of the three technical replicates for each sample using the web-based comprehensive tool RefFinder²⁷. This web-tool includes four different statistical algorithms:

The geNorm algorithm calculates the geNorm M value though a geometric averaging of all the reference genes used in the study and the mean pairwise variation of a reference gene from other genes evaluated¹⁶. Lower M values indicate greater stability: values below M ≤ 1.0 are recommended by geNorm. Good reference genes have an M < 0.5, whereas M values up to 1 are acceptable for heterogeneous samples⁵⁹;

NormFinder classifies candidate genes according to the calculation of the SV by considering intra- and intergroup variations for candidate reference genes²². We used the original NormFinder (Excel Add-in), version 0.953, to compare the ranking of the genes to the NormFinder algorithm within RefFinder web-tool. The NormFinder (Excel Add-in) is available at https://moma.dk/normfinder-software;

BestKeeper determines the stability of candidate reference genes based on SD and coefficient variation (CV) of their raw Ct values, and any gene with SD > 1 is considered unstable²⁵;

comparative delta Ct calculates the relative expression between the candidate and other genes within the same sample and ranks them based on the reproducibility of expression variation among the study samples²⁶.

RefFinder ranks candidate reference genes by integrating the results of the four programs. This tool allocates an appropriate weight to each gene and then calculates the geometric mean of their weights to obtain an overall ranking.