2.4. Real‐time PCR

The brain was taken out and the tissues of the parietal cortex, hippocampus and amygdala were obtained under an anatomical microscope. The tissues were processed for the extraction of total RNA (RNeasy Mini Kit; Qiagen, CA). The TaqmanW Universal PCR Master Mix was used to conduct RT‐PCR. For the mRNA amplification, this mix has AmpliTaq GoldW DNA Polymerase, AmpEraseW UNG, ROX passive reference, buffer and dNTPs, and gene‐specific primers. 18s rRNA was also used as an endogenous control to correct for variations in the samples. RT‐PCR was conducted in duplicate in 96‐well plates containing 2 μL of cDNA. The thermal conditions of the cycles were 50°C for 2 minutes, 60°C for 30 minutes and 95°C for 5 minutes and this procedure was followed by 40 cycles at 94°C for 20 seconds and 62°C for 60 seconds. The data were obtained in the ABI PRISM SDS 7000 thermal cycler. The 2^−ΔΔCt comparative method was used to obtain relative quantification of target gene expression and the threshold cycle value was determined by the point at which there was a statistically significant amplification in fluorescence.