Dexamethasone-Induced Gene Expression in Amygdala and NAc

Male and female mice were injected with dexamethasone (5 mg/kg; i.p.). Three hours later amygdala and NAc punches were collected for qPCR analysis. Total cellular RNA was extracted from amygdala and NAc punches using Qiazol reagent and the RNAeasy Midi kit (QIAGEN, Valencia, CA, United States). Total RNA was reverse transcribed into cDNA using VILO master mix (Invitrogen, Carlsbad, CA, United States). qPCR was performed in triplicate aliquots from each individual animal with Power SYBR Green PCR master mix (Thermo Fisher Scientific, Waltham, MA, United States), 25 ng of cDNA and 0.5 μM of primers using an ABI Prism 7900HT (Thermo Fisher Scientific, Waltham, MA, United States) in the qPCR CoRE at Icahn School of Medicine at Mount Sinai. Primer sequences for GPR83, proSAAS and GAPDH are the same as used previously (Fakira et al., 2019 (link)). The primer sequences used for qPCR are: GAPDH Forward: 5′-TGAAGGTCGGTGTGAACG Reverse: 5′-CAATCTCCACTTTGCCACTG, GPR83 Forward: 5′-GCAGTGAGATGCTTGGGTTC Reverse: 5′-CCCACCAAT AGTATGGCTCA, and proSAAS: Forward: 5′-AGTGTATG ATGATGGCCC Reverse: 5′-CCCTAGCAAGTACCTCAG. The CT values for the technical replicates were averaged and analysis performed using the ΔΔC_T method and normalized to saline controls. In some cases, qPCR reactions were repeated to determine the reliability of the primers and RNA samples.