Rna extracting solution

RNA extracting solution (Servicebio, China) was used to extract total RNA from transfected cells. Quantitative real-time PCR (qRT-PCR) was performed according to Taq Pro Universal SYBR qPCR Master Mix kit (Vazyme, China) and miRNA Universal SYBR qPCR Master Mix kit (Vazyme, China). Relative sequences of the primers were listed in Table S1. β-actin or U6 small nuclear RNA was applied as internal control.

The total RNA of the hippocampal samples was extracted using an RNA extracting solution under the guidance of the manufacturer’s instructions (Servicebio technology, Wuhan, China). RNA quality was measured using an ultramicro spectrophotometer (NanoDrop 2000, Thermo, United States). Then, RNA was reverse-transcribed into cDNA by Wuhan servicebio technology CO,. Ltd. (Wuhan, China). A quantitative-PCR analysis was performed via 2 × SYBR Green qPCR Master Mix (Servicebio technology, Wuhan, China) according to the following steps: 1) initial denaturation for 10 min at 95°C; 2) denaturation at 95°C for 15 s, anneal/extension at 60°C for 1 min for 40 cycles (Sun et al., 2020 (link)). The sequences of the primers for GFAP, BDNF, TLR4, NF-κB, TNF-α, and GAPDH are displayed in Table 1. GAPDH was selected as the internal control to homogenize the amount of cDNA in different samples. Finally, the relative mRNA expression levels were analyzed using the 2- △△Ct method.