Quantitative Real-Time PCR Analysis of Gene Expression

Quantitative real-time PCR was performed, as previously described [6 (link), 30 (link)]. Briefly, 2 μg ribonucleic acid (RNA) extracted from the amygdala was reverse-transcribed using a PrimeScript^® RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara), following the manufacturer’s instructions. PCR reactions were performed using RealMasterMix (SYBR Green) (Tiangen, China) using the a Mx3005P quantitative PCR system (Stratagene, La Jolla, CA, USA). Thermal cycling conditions were: 95 °C for 2 min followed by 40 cycles of 95 °C for 20 s, 60 °C for 20 s, and 68 °C for 40 s. To exclude the interference of unspecific products, a melting curve analysis was conducted using high-resolution data collection during an incremental temperature change from 60 to 95 °C with a ramp rate of 0.2 °C /s. β-actin was chosen as a reference. Primer sequences were: β-actin, forward-TCCATCATGAAGTGTGACGT, reverse-GAGCAATGATCTTGATCTTCAT; ERα, forward-GCTGGCCTGACTCTGCA, reverse-TCTGGCTGGGCTCCTCT; ERβ, forward-TTGCTCCAGACCTCGTT, reverse-CATCTGTCACTGCGTTCA; OTR, forward-CGTCAATGCGCCCAAAG, reverse-CGAGCAGAGCAGCAGAGGAA. Data were calculated using the 2^-ΔΔC_T formula.