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.