*2.5. Candidate Reference Gene Expression Stability Analysis*

CT values were used to assess the expression levels of candidate reference genes in all samples by RT-qPCR. Four common algorithms, namely, delta Ct (ΔCt), geNorm (version 3.5), NormFinder (version 0.953) and BestKeeper (version 1.0), were used to evaluate the stability of the expression of the candidate reference genes in the different experimental groups. In geNorm and NormFinder, the M value reflects the stability of each candidate reference gene [28,29], with a smaller M value indicating higher stability. The geNorm package also determines the number of optimal reference genes based on the ratio Vn/n+1 by calculating pairwise variations in the normalized factor after introducing a new internal reference gene [28]. A cut-off value of 0.15 was used for pairwise variation. If

the value of Vn/n+1 was less than 0.15, n was selected for the number of optimal internal reference genes; if the value of Vn/n+1 was greater than 0.15, n + 1 was selected. For BestKeeper, the values of CV and SD were used to evaluate the relative expression stability of each candidate gene [29]. The smaller the CV and SD values are, the more stable the gene. Finally, the RefFinder program (http://blooge.cn/RefFinder/, accessed on 25 September, 2022) was used to comprehensively rank the candidate reference genes by ΔCt, geNorm, NormFinder, and BestKeeper as previously described [31].
