Quantitative real-time PCR (RT-qPCR)
Total RNA was extracted from BV2 microglial cells or mesencephalic tissue using Trizol reagent (Sigma-Aldrich, USA). The RNA was subsequently reverse-transcribed into cDNA using a PrimeScript RT reagent kit (Takara, Japan). RT-qPCR was conducted using a 7300 Plus Real-time PCR System (Thermo Fisher, USA) and an SYBR Green kit (Takara, Japan). Thermocycling was conducted under the following conditions: Denaturation at 95◦C for 15 s, 40 cycles at 95◦C for 10 s, 60◦C for 30 s. The comparative threshold (Ct) method was used to analyze the data. Each 20μl reaction mixture contained 10μl PCR mixture, 1μl cDNA template, 5 pmol primer, and a proper amount of water. Primers used for RT-qPCR are outlined in Table 1.