Real-Time quantitative RT-PCR (qRT-PCR) analysis was used to confirm the direction and magnitude of mRNA levels derived from array analysis for selected genes. A preliminary experiment detailed in Methods, identified an appropriate endogenous control gene, Beta-2 microglobulin, which displayed minimal variations in message levels between treatment groups. Genes were selected from 2 relevant groups, Wnt signaling, (Lrp5, β-catenin, Dkk1) and Integrin signaling, (Itgb3). One gene (OPG) modulated by ibandronate was also selected. The selected genes all showed significant differential expression after either acute or chronic binge alcohol treatment compared to matched control values by gene array. After performing each qRT-PCR reaction, expression data for each gene was calculated for the relevant treatment groups using the 2−ΔΔCT relative method. The RNA level data for each gene produced by both gene array and PCR analysis was plotted on a shared axis as a line graph. As seen in Fig. 8, there was excellent overall correlation between gene array and qRT-PCR with respect to the direction and magnitude of gene expression across treatment groups for each selected gene.
