Real-time RT-PCR

Hippocampal BDNF and hypothalamic CRH mRNA expression levels were measured using real-time RT-PCR. Control mouse hippocampus and hypothalamus was excised 30 min after i.p. administration of YL and was kept in RNAlater RNA Stabilization Reagent (QIAGEN Sciences Inc., Germantown, MD, USA) after decapitation under deep anesthesia until RNA extraction. Total RNA was extracted from the hippocampus using the RNeasy Lipid Tissue Kit (QIAGEN Sciences Inc.) and transcribed to cDNA with random primers using Takara PrimeScript® RT Master Mix (Takara, Osaka, Japan). For quantitative PCR, we amplified the cDNA using the Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with Platinum SYBR Green qPCR SuperMix-UDG and ROX solution (Invitrogen) and each primer set specific for mouse BDNF and CRH, according to the manufacturer’s instructions. The following primers were used for real-time RT-PCR: forward bdnf: 5′-GCG GCA GAT AAA AAG ACT GC-3′, reverse bdnf: 5′-TCA GTT GGC CTT TGG ATA CC-3′, forward crh: 5′-GCA GTT AGC TCA GCA AGC TCA C-3′, reverse crh: 5′-CAA ATG ATA TCG GAG CTG CG-3′, forward β-actin: CTG CGC AAG TTA GGT TTT GTC A, reverse β-actin: TGC TTC TAG GCG GAC TCT TAC TG. The reactions were cycled 40 times with denaturation at 95 °C for 15 s, and with annealing and elongation at 60 °C for 30 s. The relative expression level of each mRNA was normalized against the mRNA expression level of β-actin.