2.5 Quantitative real-time PCR (qRT–PCR) analysis
Total RNA was extracted with a two-step isocyanate method. The total RNA
concentration was measured by an ultraviolet spectrometer, and the
OD260/280 value ranged from 1.8 to 2.0. 5 μg of total RNA was used for
reverse transcription to synthesize cDNA, and 3 μg of cDNA was used for
PCR amplification. β-Actin was regarded as an internal reference.
Primers for β-actin, TNF-α, IFN-γ, IL-1β, MCP1, MIP2, and KC were
synthesized by Primer Express. The PCR conditions included
predenaturation at 94 °C for 2 min, denaturation at 94 °C for 30 s,
annealing at 61 °C for 45 s, and extension at 72 °C for 1 min. The cycle
was repeated 36 times, and each cycle included a reduction in annealing
temperature by 0.3 °C, with a final extension of 6 min at 72 °C. The
sequences of the primers used are listed in Table 2.