Abi prism steponeplus sequence detection system

The hippocampus and PVN were collected from frozen brain slices (2 mm thick), and then stored at −80°C until assay. Total RNA was extracted from the PVN and hippocampus using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. One μg RNA was used for reverse transcription (TaKaRa, Shiga, Japan). The primer sequences were: rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH; NM_017008.4) sense: 5′-TCCCTCAAGATTGTCAGCAA-3′, and antisense: 5′-AGATCCACAACGGATACATT-3′; rat corticotropin-releasing hormone (CRH; NM_031019.1) sense: 5′-CAGAACAACAGTGCGGGCTCA-3′, and antisense: 5′-AAGGCAGACAGGGCGACAGAG-3′; rat MR (NM_013131.1) sense: 5′-TGTCTCAGACCTTGGAGCGTT-3′, and antisense: 5′-TGTTCGGAATAGCACCGGAA-3′; rat GR (AB115420.1) sense: 5′-ACAGCTCACCCCTACCTTGGT-3′, and antisense: 5′-CTTGACGCCCACCTAACATGT-3′. RT-PCR was performed using a Light Cycler Fast Start DNA Master SYBR Green I kit and ABI Prism Step One Plus Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and relative expression of genes was determined using the 2^-ΔΔCt method by normalization against GAPDH expression. The relative levels of CRH, MR and GR mRNA were expressed as percent of control value.