Lightcycler 480ii analyzer

Total RNAs of hippocampal tissues were extracted using the Trizol Reagent (Invitrogen, United States) and reverse transcribed into complementary DNA using a cDNA Synthesis Kit (GeneCopoeia, United States) according to the manufacturer's instructions. RT-qPCR was performed with the mRNA qPCR mix (GeneCopoeia, United States) accordingly. The primers (Table 1) for all assayed genes were determined utilizing revealed sequences as listed. The annealing temperature was 60°C, and the reaction was conducted utilizing LightCycler®480 II analyzer (Roche, Mannheim, Germany).

The TaqMan software was used to design the real-time quantitative polymerase chain reaction (RT-qPCR) based on exon junctions to prevent co-amplification of genomic DNA. Total mRNA was extracted using an RNA extraction kit (Omega Bio-Tek, Norcross, GA, USA) and reverse-transcribed into cDNA using a PrimeScript RT reagent kit with a gDNA eraser (Takara, Kusatsu, Japan). The primers used for the genes indicated were designed using Primer Blast. The primer sequences are listed in Tables 1, 2. The reaction was performed using a LightCycler^® 480II analyzer (Roche, Mannheim, Germany), and the amplifications were carried out for 35 cycles. β-Actin was used as the internal control, and the 2^−ΔCt method was used to calculate the differences in mRNA transcript levels.

qPCR assay was to assess the content of critical proinflammatory cytokines TNF-α, IL-α and IL-1β, AMPAR subunit GluA1 and GluA2 as well as NMDAR subunit NR2B in the hippocampus. Total RNA was isolated from homogenized hippocampal tissues using the Trizol extraction method according to the manufacturer’s instructions (Invitrogen, Waltham, MA, USA). The concentration and quality of RNA was evaluated by comparison of optical density value with NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The extracted RNA was converted into cDNA using cDNA Synthesis Kit (GeneCopoeia, Rockville, MD, USA) under GeneAmp^®PCR system 9700 (Applied Biosystems, Carlsbad, CA, USA). qPCR was performed using mRNA QPCR mix (GeneCopoeia, Rockville, MD, USA) under LightCycler^®480II analyzer (Roche, Mannheim, Germany). The primers for all assayed genes were determined using reported sequences as listed in Table 1. The qPCR reaction was run at 95°C for 10 min and followed by a repeating 40 cycles of denaturation at 95°C for 10 s, primer annealing at 60°C for 20 s and an extension at 72°C for 20 s, terminated by heating to 72°C for 5 min. After amplification, the gene expression changes were quantified and analyzed using the 2^−ΔΔCt method for all samples. The results were normalized to GAPDH as reference gene.