The hippocampus was homogenized using the TissueLyser LT magnetic bead homogenizer (Qiagen, Hilden, Germany) with 50-mm stainless-steel beads for 5 min at 50 Hz. Total RNA was isolated using TRIzol reagent (Life Technologies, Carlsbad, CA, USA), and cDNA was synthesized from RNA using the M-MLV RT reaction buffer (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The cDNA was mixed with forward and reverse primers for Bdnf coding (forward: GCGGCA-GATAAAAAGACTGC; reverse: GCAGCCTTCCTTGGTGTAAC), and Bdnf promoter (forward: GCCTTCATGCAACCGAA-GTA; reverse: CAGGACAGCAAAGCCACAAT) (Sleiman et al., 2016 (link)) in a 96-well plate containing AMPIGENE qPCR Green Mix Lo-ROX (Enzo Life Science, Farmingdale, NY, USA). To normalize mRNA levels among samples, Actb (forward: CCAACC-GTGAAAAGATGACC; reverse: CCATCACAATGCCTGTGGTA) was used as a housekeeping gene. Each sample was examined in triplicate. Sequences of all primers were entered 5′ to 3′ order. The reaction was performed using a LightCycler 96 real-time PCR system (Roche Diagnostics, Basel, Switzerland). The cycle threshold (Ct) values measured after the experiment were analyzed by the relative quantification 2−ΔΔC(T) method.