cDNA was analyzed by quantitative real-time polymerase chain reaction using an Applied Biosystems 7500 instrument (Thermo Fisher Scientific) and TaqMan Assay On Demand (Thermo Fisher Scientific) FAM-labeled probe and primer sets for: GABRA1 (hs00168058_m1), GABRG2 (hs00158093_m1), and GABRD (hs00181309_m1). Expression of these genes was normalized to the VIC-labeled housekeeping gene GUSB (4326320E), which we used due to its moderate abundance in our culture system and our prior demonstration of its reliability in studies of alcohol and genetic variation in iPSC-derived neural cells (Lieberman et al. 2012, Lieberman et al. 2015). cDNA synthesized from RNA extracted from each culture well was assayed in triplicate 20 μL reactions using Gene Expression Master Mix (Thermo Fisher Scientific) per the manufacturer’s protocol.
