RNA was extracted from the cortex brain area using Trizol according to the manufacturer’s instructions (Sigma). RNA was measured in a NanoDrop ND-1000 Spectrophotometer (260/280 nm ratio). First-strand cDNA synthesis was performed with the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Biosystems) using 2000 ng of total RNA according to the manufacturer’s instructions. The RT-PCR reactions contained LightCycler 480 SYBR Green I Master (2×; Roche Applied Science), 5 μM forward and reverse primers, and 1 μl of cDNA. RT-PCR was performed in a LightCycler® 480 System (Roche). The amplification efficiency (E) of the primers was calculated from the plot of the Cq values against the cDNA input according to the equation E = [10(-1/slope)]. The relative expression ratio of a target/reference gene was calculated according to the Pfaffl equation (Pfaffl, 2001). The sequence of both the forward and reverse primers used in this study is detailed in Table 1. Housekeeping cyclophilin A (PPIA) was used as an internal control. Fluorescence was recorded in the annealing/elongation step in each cycle. A melting curve analysis was performed at the end of each PCR to check the specificity of the primers.
