Hippocampal Gene Expression Analysis

RNA extraction, cDNA synthesis, and RT-qPCR were performed as described previously (Weerasinghe-Mudiyanselage et al., 2022b (link)). RNA extraction from hippocampal tissue (n=5/group) was conducted using TRIzol reagent according to the manufacturer’s instructions (#74106, Qiagen, Germany). Reverse transcription was performed using a Superscript™ III cDNA Synthesis Kit (#EZ405S, Enzynomics, South Korea). The resulting cDNA was diluted with RNase-free water to achieve a final concentration of 8 ng/µL, with the samples then stored at −70°C. RT-qPCR was performed using TOPreal^TM SYBR Green qPCR PreMix (#RT500M, Enzynomics, South Korea) and the LineGene 9600 Plus system (BIOER, China) following the manufacturer’s instructions. All RT-qPCR primers are listed in Table 1. The annealing temperature for the reaction was 58°C, and the built-in software generated the amplification curves and threshold cycle values. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as the reference gene to normalize all readings. Data were expressed as mean relative values compared to the values of the control group via the 2−^∆∆CT method (Livak & Schmittgen, 2001 (link)).