2.9 RT-PCR
Total RNA was extracted from mouse brains and cells using the Trizol reagent (Sigma Aldrich, USA). The RNA concentration was quantified using spectrophotometry with a spectrophotometer (Thermo Fisher Scientific, Waltham, USA). The Reverse Transcription Master Mix (Takara, Tokyo, Japan) was utilized to synthesize cDNA by reverse transcription at 42℃ for 15 minutes followed by a denaturation step at 85℃ for 5 seconds. The mRNA expression was analyzed using SYBR Premix Ex Taq TM II (Takara, Tokyo, Japan) and synthetic primers on a thermocycler (Bio-Rad, Hercules, California, USA). The relative mRNA expression levels were calculated and quantified with the 2−ΔΔCt method after normalization to reference GAPDH expression42. The primers utilized for quantitative real-time PCR in this investigation are enumerated in Table 1.