2.3.1. RNA Isolation, cDNA Synthesis, and Quantitative Real-Time-PCR (qPCR)

Total RNA was extracted individually from all the brain tissues of each mouse using Trizol^® Reagent (Invitrogen, USA) according to the manufacturer’s protocol. RNA quality control was performed by Nanodrop where the ratios 260/230 and 260/280 were always around 2.00. After RNA extraction, DNase treatment with RNase-free DNase I (Invitrogen, Waltham, MA, USA) was applied according to the manufacturer’s protocol to prepare DNA free RNA. For the first strand cDNA synthesis, three micrograms of total RNA of each sample was used with random hexamers (Applied Biosystems, Bedford, MA, USA) and SuperScript™ III Reverse Transcriptase (Invitrogen, Waltham, MA, USA) in cDNA synthesis. In qPCR, every reaction was made in four parallel samples to minimize possible errors. All reactions were performed in a final volume of 10 μL, using 5 ng of cDNA. Real-time qPCR was performed using 5x HOT FIREPol^® EvaGreen^® qPCR Supermix (Solis BioDyne, Tartu, Estonia). For thermal cycling, a QuantStudio 12 K Flex Software v.1.2.2 Real-Time PCR System equipment (Applied Biosystems, Bedford, MA, USA) was used at 95 °C for 15 min, then 40 cycles at 95 °C for 20 s, 61 °C for 20 s and 72 °C for 20 s. All qPCR data has been presented in the log₁₀ scale, in the form of 2^−ΔCT, where ΔCT is the difference in cycle threshold (CT) between the gene of interest and housekeeper gene hypoxanthine guanine phosphoribosyl transferase (Hprt). For quality control, three internal samples with Hprt primers were used between all the measurements.