Quantification of gene expression by real time PCR

Total RNA from VMB neurons or rat substantia nigra were isolated using the RNeasy Mini Kit from Qiagen (Valencia, CA) and 1μg of total RNA was reverse transcribed using SuperScript III first-strand synthesis supermix for qRT-PCR from Invitrogen (Carlsbad, CA) according to the manufacturer's instruction. Quantitative RT-PCR was performed using Maxima SYBR Green master mix (Fermentas) in ABI Prism 7900HT in four replicates with primers amplifying the target genes. Expression level of actin was used as an internal control. Data were normalized using the 2^−ΔΔCt method [43]. Levels of target gene mRNAs were expressed relative to those in vehicle control groups and plotted in GraphPad Prism.