Allelic mRNA expression level is determined by comparing the number of genomic DNA molecules for each allele with the number of allelic mRNA molecules within the same individual and same tissue, thus mRNA expression levels from the paternal and maternal alleles would be the same unless there are cis-regulatory variants that affect gene expression. We selected subjects with heterozygous genotypes for the coding SNP (rs16969968) and/or a SNP (rs615470) in the 3′UTR of CHRNA5 for allele-specific expression measures. TaqMan genotyping assays for rs16969968 (Life technologies, C_26000428_20) and rs615470 (Life technologies, C_18757_10) are within the CHRNA5 transcript, and were used for allele specific quantitative RT-PCR in each genomic DNA and cDNA. To enhance RT-PCR template, we pre-amplified gDNA and cDNA using primers flanking the region targeted by the TaqMan genotyping assay and ran a standard PCR reaction for 20 cycles. Primers used for rs16969968 pre-amplification are: forward primer 5′-CGCCTTTGGTCCGCAAGATA-3′ and reverse primer 5′-TGCTGATGGGGGAAGGTGGAG-3′. Primers used for rs615470 pre-amplification are: forward primer 5′- CAGATGATCCATTTGAACAGTTGGC-3′ and reverse primer 5′- TAGAGACGGGGTTTCTCCACGTTG-3′. Portions of pre-amplified cDNA from heterozygous samples for rs16969968 and/or rs615470 were arrayed
