Five different approaches were employed to determine the expression stability of the candidate reference genes. The geNorm, or pairwise comparison approach, ranks candidate genes according to their expression stability [27]. The gene stability measure, or M, is determined for a given reference gene as the average pairwise variation for that gene in relation to the remaining tested reference genes. The gene with the highest M is stepwise excluded, with a new M value being repeatedly calculated for the remaining genes until a final pair of genes with the lowest M (the most stable) are obtained. Moreover, this Excel-based software determines the optimal number of references genes required for an accurate normalization of real-time PCR data. For this, the pairwise variation (Vn/Vn+1) was analyzed between the normalization factors NFn and NFn+1; Vn values were calculated by stepwise inclusion of additional reference genes until the (n+1) gene did not contribute significantly to the newly determined normalization factor. NormFinder software (also Excel-based) is a model-based approach that ranks the candidate reference genes according to their minimal combined inter- and intra-group expression variation [28]. Such variations are combined to give a stability value, with the lowest value corresponding to the most stable expression. BestKeeper (an Excel-based statistical method) calculates the standard deviation (SD) and coefficient of variation (CV) from the input raw CT values, with genes exhibiting the lowest SD and CV being considered as the most stable [29]. Reference genes with SD higher than 1 are considered as inconsistent and are excluded. The comparative ΔCT algorithm uses the average SD values to rank the stability of all candidate reference genes, and the reference gene with the lowest SD is considered as the most stable [30]. Finally, as a complementary analysis to assess reference gene stability, the web-based tool RefFinder was employed to integrate the results from the four previous software packages, generating a comprehensive rank list of candidate genes [31].

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