Hepatic hepcidin and Tfr1 mRNA expression in weanling and adult mice of both sexes and genotypes, and in pregnant mice of both genotypes. qRT-PCR was performed to assess hepcidin (A-C) and Tfr1 (D-F) mRNA expression levels in liver samples from adult (A,D), weanling (B,E), and pregnant or nonpregnant female (C,F) mice. Homogeneity of variances was assessed by Levene’s test. Because there was no homogeneity of variance in these data sets (except for Tfr1 expression levels in adults), data were log10 transformed prior to statistical analysis. For ease of interpretation, however, the nontransformed data are shown in the figure. Data are presented as box-and-whisker plots and were analyzed by 2-way ANOVA. Data from experimental genes were normalized to the expression of Rps18. A significant genotype main effect was noted for hepcidin mRNA expression in adult mice (P = .0011) (A). Sex (P = .0013) and genotype (P < .0001) main effects were noted for hepcidin mRNA expression in weanling mice (B). A significant 2-way interaction was noted for pregnancy × genotype (P = .0009), and significant sex (P < .0001) and genotype (P = .0001) main effects were additionally noted for hepcidin mRNA expression in pregnant mice (as compared to nonpregnant females of a similar age) (C). Because a significant 2-way interaction was noted, multiple pairwise comparisons were made by Tukey's HSD post-hoc test; letters atop bars indicate statistically significant differences (P < .05). Tfr1 expression data from adult mice (D) revealed no significant main effects or interactions. In weanling mice, significant sex (P = .0279) and genotype main effects (P < .0001) were noted in regards to Tfr1 mRNA expression (E). The main effects of pregnancy (P = .0001) and genotype (P = .0053) on Tfr1 mRNA expression were significant when pregnant mice were compared to nonpregnant female mice (F). n = 5-6 mice per group, except for the adult, control female group, which had 3 mice, and the weanling, KO female group, which had 4 mice. Each PCR reaction was run in duplicate and Ct values were averaged.
