Real-time PCR for inflammatory mediators

Gene expression of MCP-1, CCR-2, TNF-α, IL-1β, and IL-6 in the lumbar spinal cord was measured using real time qPCR. Total RNA of lumbar spinal cords from vehicle or Mac-1-saporin treated groups was extracted using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Total RNA (1 μg) was used as template for reverse transcription. Real-time quantitative PCR reactions (in triplicate) were processed with a Rotor-Gene Q real-time PCR cycler (Qiagen) using SYBR Green Supermix from BIO-RAD. GAPDH was used as the internal control. The sequences of primers used in Real-Time PCR were listed in Table 1.