Neural cell culture cDNA was analyzed by real time polymerase chain reaction (RT-PCR) using an Applied Biosystems 7500 instrument and TaqMAN Assay on Demand (Applied Biosystems) probes and primers for: GRIN1 (Hs00609557_m1), GRIN2A (Hs00168219_m1), GRIN2B (Hs00168230_m1), and GRIN2D (Hs00181352_m1). Expression of these genes was normalized relative to the expression of the housekeeping gene GUSB (Applied Biosystems, 4326320E) co-amplified with target genes. PCR cycles were as follows: 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. A standard curve consisting of a 4-level 2-fold serial dilution series of pooled cDNA taken from all samples in the experiment was used to determine relative mRNA expression levels. Expression after 7 days of alcohol exposure and 7 days of alcohol exposure plus a 24-hour withdrawal period was normalized to each subject’s sham condition. Data were analyzed for statistical significance with mixed model repeated measures test using SPSS software.
