Optimized Real-time PCR for Brain Gene Expression

Primers for avp were designed and optimised by Primerdesign Ltd. The primer sequences were: avp forward: 5′-CTGCCTGCTACATCCAGAACT-3′, avp reverse: 5′-CACACGACATACACTGTCTGATG-3′. The sequences of the primers for oxt, th, th2, tph1a, tph1b, tph2, avpr1aa, avpr1ab were taken from^{33 (link)} and purchased from Sigma. RNA was extracted from the whole brain using the GeneElute^TM Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) followed by a DNase treatment with Turbo DNase (Ambion). The quality and quantity of RNA was assessed using a Nanodrop 2000 (Thermo Scientific). cDNA was synthesised from 0.5 µg of RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). Real-time PCR was performed on 8 whole brains per genotype with three replicates for each brain using a CFX Connect^TM Real-Time System machine (BIORAD) and the SensiFAST^TM SYBR No-ROX Mix (Bioline). The PCR conditions were 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 15 s and 72 °C for 30 s. Results were normalised to the expression level of the housekeeping gene rpl13. The relative expression of the genes was calculated using the comparative 2^−ΔΔCt method as described in^{97 (link)}.