2.5 RNA extraction and gene expression determination
Total RNA isolation from hippocampal samples was performed using the TRIzol® reagent according to the manufacturer’s instructions (Bioline Reagent). The yield, purity and quality of RNA were determined spectrophotometrically with a NanoDrop™ND-1000 apparatus (Thermo Scientific) and an Agilent 2100B Bioanalyzer (Agilent Technologies). RNA samples with 260/280 ratios and RINs higher than 1.9 and 7.5, respectively, were selected. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed. Briefly, 2 μg of messenger RNA (mRNA) was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems).
SYBR® Green real-time PCR was performed using a Step One Plus Detection System (Applied-Biosystems) with SYBR® Green PCR Master Mix (Applied-Biosystems). Each reaction mixture contained 6.75 μL of complementary DNA (cDNA) (with a concentration of 2μg), 0.75 μL of each primer (with a concentration of 100nM), and 6.75 μL of SYBR® Green PCR Master Mix (2x).
The data were analysed utilising the comparative cycle threshold (Ct) (ΔΔCt) method, in which the levels of a housekeeping gene are used to normalize differences in sample loading and preparation. Normalization of expression levels was performed with β-actin. The primer sequences and TaqMan probes used in this study are presented in Supplementary Table S2. Each sample was analyzed in duplicate, and the results represent the n-fold difference in the transcript levels among different groups.