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.