2.7. RT-PCR Cytokine Expression

Brains were extracted and placed in RNAlater solution for storage before RNA extraction. The brain tissue was weighed and then homogenized in 1 mL TRIzol per 100 mg tissue. Separation of DNA and proteins was then performed using 200 μL chloroform per 100 mg tissue and centrifugation. Once the RNA was isolated, a series of wash steps using isopropyl alcohol and ethanol was performed to precipitate pure RNA. The purity of the RNA samples was assessed using Nano Drop (ND-1000, Thermo Fisher, Waltham, MA, USA). Reverse transcription was performed using 2500 ng total RNA and 4 μL of reagent (SuperScript™ IV VILO™ (SSIV VILO) Master Mix, Thermo Fisher, Waltham, MA, USA). Primers were annealed for 10 min at 25 °C, incubated for 10 min at 50 °C, and then at 95 °C for 5 min. The volume of cDNA was diluted to obtain 100 μL of 20 ng/μL cDNA. Once the cDNA was isolated, qRT-PCR was performed with Fast SYBR Green Master Mix (Thermo Fisher, Waltham, MA, USA) by a StepOnePlus RealTime PCR System. iTaq Supermix, cDNA, and forward/reverse primers for each respective cytokine were prepared and kept on ice with a total reaction mix volume of 10μL including 20 ng of cDNA. The reactions were loaded into triplicate wells and loaded into a CFX RT-PCR system. The expression of GAPDH mRNA was used to normalize target gene expression levels, and the P14 age group of mGluR5+/+ was used as the control for comparative cycle threshold (2^−ΔΔCt) calculations. All gene expression levels were normalized and are expressed as the relative fold change to the housekeeping gene of the control condition.