Trizol

Cell lysates were homogenized in 1 mL Trizol (Invitrogen, Waltham, MA, USA) and incubated at room temperature for 10 min. 0.2 mL chloroform (Chem Supply, Melbourne, Australia) per 1 mL Trizol was added to the samples and they were centrifuged at 12,000× g for 15 min at 4 °C. The colorless, aqueous phase of each sample containing the RNA was transferred into a fresh 1.7 mL microcentrifuge tube. RNA was precipitated by adding 0.5 mL Propan-2-ol (Chem Supply) per 1 mL Trizol and the samples were centrifuged again at 12,000× g for 10 min at 4 °C. The supernatant from the tubes was discarded, and the RNA pellet was washed with 75% ethanol (Chem Supply, Melbourne, Australia) in diethyl pyrocarbonate (DEPC)-treated water (Sigma, St. Louis, MO, USA), vortexed and centrifuged at 7500× g for 5 min at 4 °C. The RNA pellet was air-dried and redissolved in RNAse-free H₂O (Invitrogen, Waltham, MA, USA). The concentration and purity of the RNA samples was measured using the NanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Cortical and striatal regions of the brain were isolated from the ipsilateral and contralateral hemispheres and were homogenised in 1 ml Trizol (Invitrogen) before incubation at room temperature for 10 min. Then, 0.2 ml Chloroform (Chem Supply) per 1 ml Trizol was added to the samples, and samples were centrifuged at 12,000 g for 15 min at 4 °C to separate samples into phases. The colourless, aqueous phase of each sample, which contained RNA, was transferred into a fresh 1.7 ml microcentrifuge tube. RNA was precipitated by adding 0.5 ml Propan-2-ol (Chem Supply) per 1 ml Trizol, and samples were again centrifuged at 12,000 g for 10 min at 4 °C. The supernatant from the tubes was discarded, and the RNA pellet was washed with 75% Ethanol (Chem Supply) in diethyl pyrocarbonate (DEPC)-treated water (Sigma), vortexed and centrifuged at 7500 g for 5 min at 4 °C. The RNA pellet was air-dried, and redissolved in RNAse-free H₂0 (Invitrogen). Concentration of the RNA samples was measured using the NanoDrop 1000 Spectrophotometer (Thermo Scientific).