Cocaine-Induced Gene Expression in Nucleus Accumbens

For biochemical experiments animals were treated with high dose cocaine (20 mg/kg/day i.p.) and killed 24 hours after the final dose of cocaine. Nucleus accumbens tissue was rapidly dissected and frozen on dry ice. RNA isolation, qPCR and analysis were done as previously described60 (link). Briefly, RNA was extracted by homogenizing tissue in Qiazol reagent (Qiagen) and purified using RNeasy micro kits from Qiagen according to manufacturer protocols. RNA concentration and quality was assessed using a NanoDrop spectrophotometer (Thermo). Reverse transcription was performed using iScript (BioRad). qPCR using SYBER green master mix (Quanta) was carried out using an Applied Biosystems 7900HT cycler with the following parameters: 2 min at 95 °C; 40 cycles of 95 °C for 15 s, 59 °C for 30 s, 72 °C for 33 s; and graded heating to 95 °C to generate dissociation curves to confirm amplification of a single PCR product. Data were analyzed by comparing C(t) values of control and antibiotic treated mice using the ∆∆C(t) method61 (link). Primer pairs used for analysis are available in Table 1.