Total RNA preparation and RT–PCR analysis
Total RNA was isolated according to the supplier's protocol (TRI Reagent; Molecular Research Center, Inc.). For RT–PCR, 2 μg of total RNA was reverse transcribed and PCRs were carried out with MOR-specific primers at the same tube using one-step RT–PCR reagent (Qiagen) in a GeneAmp 9600 PCR machine (Perkin-Elmer). The PCR cycle conditions for MOR consisted of 95°C for 45 s, 60°C for 45 s and 72°C for 45 s, followed by a 10 min extension at 72°C (37 cycles for NS20Y, 33 cycles for PC12 cell). PCR products were separated in a 2.0% agarose gel with TBE buffer. The mouse MOR transcript was amplified with primer set P2Ss (5′-CTCCGTGTACTTCTAAGGTGGGAG-3′) and TM2as (5′-GGCTAAGGCATCTGCCAGAGCAAG-3′). Similar reactions were carried out using primers for β-actin as an internal control. Quantitative analyses were carried out using ImageQuant 5.2 (Amersham) software.