μl 0.1mol/l dithiothreitol, 1 μl RNasin, 2 μl SSII (50 u/μl), 5 μl hexomers and RNase-free water to 40 μl. Incubation was then carried out at 42°C for 60 min followed by heat inactivation at 70°C. Finally, 1 μl of RNase H was added to each reaction and incubated at 37°C for 20 min to degrade the RNA. For real-time quantitative PCR, reactions were conducted in a volume of 4 μl using the Sybr Green I master kit (PE applied Biosystems, Foster City, California, USA). Briefly, 2 μl of a mixture of 2x Sybr Green and primers were loaded with 2 μl diluted cDNA template in each well. Following this, 8 μl of mineral oil was loaded in each well to prevent loss of solution. Using an ABI prism 7900HT system (Applied Biosystems, Foster City, California, USA), PCR was carried out using the parameters 52°C, 5 min→95°C, 10 min then (95°C, 30s→60°C, 60s) for 40 cycles. Samples were analyzed in triplicate. Melting curves were performed to document single product formation, and agarose electrophoresis confirmed appropriate product size. For internal control 18s RNA was used. The 18s primers were purchased from Ambion (Austin, Texas, USA). The expression of Htr3a in morphine-treated
