to an average CT value from two housekeeping genes (Gusb and Hprt). Although Gapdh, β-actin, and Hsp90ab were also on the array and available for use as housekeeping genes, our lab has found that prenatal alcohol exposure impacts the expression of these genes (unpublished data); thus, we chose to use only average CT values from Gusb and Hprt as normalizing housekeeping genes. The comparative CT method (ΔΔCT) was used to assess changes in mRNA levels. For each gene, the fold change comparing prenatal alcohol exposure (PAE) and control conditions was determined: fold change was calculated as 2-ΔCT(PAE)/2-ΔCT(control). Relative fold-change expression was calculated as 2-ΔCT(PAE sample)/2-ΔCT(AVG control); all graphs indicate relative fold change comparing PAE to control gene expression. All control and PAE tissue was age-matched to avoid confounds. Results from the microarrays were validated using qRT-PCR in triplicate using new sets of cell cultures from different dams. RNA (1 μg) was isolated from cells as described above and stored at −80 °C until cDNA conversion. Reverse transcription was performed using a Quantitect Reverse Transcription Kit (Qiagen; 205311) and a Peltier Thermal Cycler. Quantitative PCR was performed using FastStart Universal SYBR Green (Roche; 04913850001) on an ABI 7300 Real-Time PCR System
