Pamm 085

Gene expression levels were determined as described ^{45 (link)}. For PCRarrays, RNA was converted to cDNA and analyzed on a mouse epigenetic chromatin modification enzymes PCR array platform (PAMM-085, SA Biosciences) following the manufacturer’s protocol. PCR arrays results were analyzed using the RT² Profiler PCR Array Data Analysis (SA Biosciences).

A RT-qPCR array containing primers for 84 chromatin modifying enzymes was used to screen EtOH-induced gene expression changes according to the manufacturer’s protocol (SA Biosciences, #PAMM-085; full gene list shown in Supplementary Table 2). RNA was extracted and purified according to the RT-qPCR section, converted to cDNA, and 8.5 ng of cDNA used per well according to the manufacturer’s protocol. A total of 6 PCR arrays were used (3 saline-treated and 3 EtOH-treated animals). qPCR conditions were 10 min at 95°C (initial denaturation) followed by 40 cycles of 15 s at 95°C (denaturation) and 1 min at 60°C (annealing and extension). Ct values from each PCR array were normalized to the median Ct value of that array (median normalization). Median normalized Ct values were further normalized to the average of saline controls (ΔΔCt) and fold change values calculated using the following formula: 2^−ΔΔCt.
Genes whose expression was changed >100% after EtOH treatment or had a p-value <0.1 and change in expression >25% were chose for validation by RT-qPCR using an additional 6 mice per group.