3.3.9. RNA Isolation and qPCR

Brain regions were isolated by placing the brain into an acrylic matrix (Able Scientific, Canning Vale, WA, AUS) and subsequently cutting them into 1 to 2 mm thick sections. The nucleus accumbens and the hypothalamus was subsequently microdissected using micropunches (Kai Medical, Seki City, Japan) and submerged in RNAlater® ICE (ThermoFisher Scientific, Waltham, MA, USA) prior to performing RNA isolation. RNA was isolated using Maxwell® 16 LEV simply RNA Tissue Kit (Promega, Madison, WI, USA) as per manufacturer instructions. RNA was quantified using spectrophotometric analysis, with the quality of RNA verified by the OD260/280 ratio. 900 ng of RNA was reversed transcribed into cDNA using iScript™ cDNA reverse transcription kit (BioRad, Hercules, CA, USA) as per manufacturer instructions.

Gene expression was assessed using iTaq™ Universal SYBR® Green Supermix (as per manufacturer instructions). Real time PCR was performed using the CFX96 Touch™ Real-Time PCR Detection System (BioRad). All primers were synthesised by Integrated DNA Technologies Pte. Ltd. (Baulkham Hills, NSW, Australia) with their sequences are outlined in the supplementary material (Table 1).

The relative difference in expression level of each of the genes of interest were normalised to the CT of GAPDH for both the test and control sample. The ΔCT of the test sample was normalised to the ΔCT a control sample (equal amount of cDNA from all samples), and then expressed as a ratio (2^−ΔΔCT).