Quantitative Real-Time PCR Analysis of Inflammatory Genes

Quantitative real time PCR was performed using TaqMan Probe single tube assays (Life Technologies) for mouse (Ccl2, Cxcl16, Arg1, Il1b, Cd36, and Mmp2) genes. The StepOnePlus Real-Time PCR System (Applied Biosystems) was used for amplification and detection. Threshold cycle number was determined using Opticon software. mRNA levels were normalized to β−actin, which control studies showed is not altered by CDDO-Me treatment, using the equation 2^−(Et-Rt), where Rt is the mean cycle threshold for the control gene and Et is the mean threshold for the experimental gene. Thermal cycling conditions for qRT-PCR consisted of an initial incubation at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec and 60 °C for 1 min. Product accumulation was measured during the extension phase and all samples were run in triplicate^{12 (link)}.