Cells grown in monolayer or dissected tissue was homogenized in RNA-STAT60 (Tel-Test Inc., Friendswood, TX) using a 27 gauge needle. Following homogenization, 250 μl of chloroform was added and the samples were vortexed for 1 min. Samples were then centrifuged for 15 min at 12,000 × g at 4°C, and the upper aqueous RNA containing layer was removed for an additional RNASTAT60/chloroform extraction. The RNA was precipitated with 2 × volume of isopropanol overnight at −20°C and centrifuged for 30 min at 12000 × g. The RNA pellets were washed twice with 70% ethanol/RNAase-free water and subsequently resuspended in RNAsecure (Ambion/Applied Biosystems, Austin, TX), and ~10 μg of RNA from each sample was treated with Turbo DNase (Ambion/Applied Biosystems) for 60 min at 37°C to degrade residual genomic DNA. To assess RNA levels, samples were reverse transcribed into cDNA with the TaqMan High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Thereafter, they were processed with the TaqMan Universal PCR kit with the mouse or rat CHRNA5 gene expression assay (Applied Biosystems); controls consisted of either β-actin or
