qRT-PCR

qRT-PCR was conducted as previously reported (Zhuang et al., 2021). Total RNA was extracted from the hippocampus using Trizol reagent according to the manufacturer’s instructions. The purity and content of total RNA were determined using a spectrophotometer. The PrimeScript™ RT reagent Kit (Takara, RR047A) was used for reverse transcription of total RNA to complementary DNA (cDNA). Transcripts were amplified by qRT-PCR using Novostart SYBR qPCR SuperMix Plus in a 10 μl total reaction mixture (5 μl of 2× SYBR Green mixture, 1 μl of each primer (10 μM), 1 μl of cDNA template, and 2 μl of RNase-free water). qRT-PCR was performed under the following conditions: one cycle of 95°C for 1 min, 40 cycles of 95°C for 20 s, and 60°C for 1 min. mRNA levels were quantified using the 2^△△Ct method. Beta-actin was used as the endogenous control, and the primer sequences are listed in Table 1.

Primer sequences used for qRT-PCR.