Total RNA was isolated using the quick RNA microprep kit (Zymo, Cat. #11-328M) per manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 100 ng total RNA and random hexamers using the qScript™ XLT cDNA SuperMix (QuantaBio, Cat. #955161-100). Quantitative real time PCR (qRT-PCR) was performed using TaqMan Probe single tube assays (Life Technologies, Cat. #4324018) for human CCL2 (Applied Biosystems, Cat. # Hs00234140), IL-6 (Cat. #Hs00985639_m1) and VEGF (Cat. # Hs00900055_m1) 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 (11 (link)), 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.
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