We used the Qiagen RNeasy and Lipid Tissue kit (Qiagen, Valencia, CA, USA) to extract total RNA from brain tissue. RNA concentration was measured with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Pittsburgh, Philadelphia, USA) and 5–10 ug total RNA was converted to cDNA with a High Capacity cDNA Archive kit (http://www.appliedbiosystems.com). TaqMan assays were used to quantify the total mRNA expression of CHRNA5 (Hs00181248_m1; Applied Biosystems, CA, USA), and PSMA4 (Hs01002583_m1) in human frontal cortices. Gene expression levels were assessed by real-time PCR using an ABI-7900HT Fast real-time PCR system. Each real-time PCR run included within-plate triplicates. Correction for sample-to-sample variation was done by simultaneously amplifying GAPDH (Hs02758991_g1) as a reference. We used the comparative Ct method to analyze total mRNA expression levels of CHRNA5 and PSMA4 and then normalized with GAPDH mRNA expression to obtain relative total mRNA expression level. A detailed protocol is described in Wang et al. [19], [20].
