A-D: Comparison of a target gene mRNA expression normalized with different subsets of internal control genes (ICGs).
NR3C1 raw RT-qPCR data were normalized to different subsets of ICGs: (A) raw RT-qPCR data geometrical mean of HPRT1, PPIA, RNA18S, and RPLPO, the most reliable normalization factor as uncovered by the use of geNorm; (B) “flat gene” = RNA18S and (C) “flat gene” = HPRT1; and (D)
ACTB as the least reliable ICGs as uncovered by using geNorm (besides being among the most popular used ICG). Data were analyzed by MANOVA with treatment, gender and day of gestation as main effect, followed by a pairwise comparison (Holm’s Sidak) when main effects were p < 0.05. Different letters indicate significant differences in day of gestation; stars indicate significant differences in DEX treatment. The final data were obtained by rescaled normalized expression: Qnormalized/rescaled = (Qsample/NFsample)/Min (Qsample/NFsample) (geNorm v3.5 manual ).