2.4.2. Brain-Derived Neurotrophic Factor (BDNF) Assessment by Reverse Transcription qPCR (RT-qPCR)

Firstly, total RNA was assessed. According to manufacturer instruction, tissue samples were homogenized in liquid nitrogen and submitted to chemical extraction by a RiboPure™ – Blood Kit (Ambion, USA) and treated with RNase-free DNase I. Total RNA concentration was determined by fluorimetry using the Qubit™ RNA BR Assay kit (Invitrogen, USA). The cDNA was immediately synthesized using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, USA). GoScript™ Reverse Transcription System (Promega Corporation) was used, following the manufacturer's instructions for cDNA synthesis. Real-time PCR (qPCR) was performed using GoTaq® Probe qPCR Master Mix (Promega Corp.), as described previously [55]. All reactions were carried out in triplicate in 96-well PCR plates, using CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, USA). Data analysis was performed employing the Bio-Rad CFX Manager™ 3.1 software (Bio-Rad). MIQE guidelines was adopted, which the expression levels were normalized by β-actin (Actb) in control samples. Then, the relative gene expression was measured applying the formula 2 − ΔΔCT (p < 0.05). The expression of Bdnf and Actb was evaluated through Taqman® gene expression assays (Applied Biosystems, USA) (Rn02531967 and Rn00667869, respectively).