We used the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, California, USA) to extract DNA from brain tissue. We used the PrimerPicker software (http://www.kbioscience.co.uk/primer-picker/) to design the assay and followed the protocol described in the KASPar SNP Genotyping System manual (http://www.kbioscience.co.uk) to run PCR reactions with an ABI GeneAmp PCR System 9700 (Applied Biosystems www.AppliedBiosystems.com). Genotypes were evaluated using an ABI instrument, 7900 HT Fast Real-Time PCR system. The 22-bp insertion/deletion variant, rs3841324 was genotyped using electrophoresis method described in our previous study [20]. We used Haploview software (v3.2) [26], to infer the LD structure of the genome in the region containing loci associated with CHRNA5 expression in brain samples tested.
