Figure 4.

Gnas expression by BPA exposure and age. Based on BPA-related DHMR annotated to the Gnas gene, real-time quantitative polymerase chain reaction (RT-qPCR) was used to investigate longitudinal blood Gnas mRNA expression levels. RNA was isolated from the same longitudinal Control ($n=6$ per age group) and BPA-exposed ($n=6$ per age group) mouse blood samples used for DNA hydroxymethylation analyses. RT-qPCR was performed on the Gnas gene in triplicate. Three housekeeping genes—$\mathrm{\beta }\text{-actin}$, 18S, and Gapdh—were included as internal controls in all RT-qPCR runs. In addition to housekeeping genes, an inter-plate calibrator control of brain cDNA was included for calculation of relative gene expression across multiple plates; all expression values are shown relative to this inter-plate calibrator. Expression levels were calculated following the $2^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method. ^aMean Gnas expression in BPA-exposed mouse blood showed a significant increase between 2 and 10 months of age ($p=0.05$); this pattern was not found in Control samples. ^bMean Gnas expression was significantly lower in Control blood than BPA-exposed blood at 10 months of age ($p=0.01$).