Quantitative Analysis of Inflammatory Markers

After 4 hours of incubation, monocytes were harvested for RNA extraction. Total RNA was isolated from cultured cells using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's directions. cDNA was prepared from 1 μg of total RNA using PrimeScript II RTase (Takara Bio, Shiga, Japan) with Oligo dT primers (Takara Bio), and was subjected to analysis with real-time PCR using LightCycler 4.1 (Roche Diagnostics, Lewes, UK). Real-time PCR for IL-6, TNF-α and β-actin was performed using SYBR Premix Ex Taq II (Takara Bio) as previously described [13 (link),15 (link)]. Amplification was performed according to the standard protocol recommended by the manufacturer. All results for the copy number of IL-6 mRNA or TNF-α mRNA were calibrated to the copy number of β-actin obtained from the same cDNA samples. Real-time PCR for NFkB1 (p50), NFkB2 (p52) and RelA (p65) in cultured monocytes was also performed as previously described [16 (link)]. The expression of mRNA for NFkB1 (p50), and RelA (p65) is shown as the ratio of the copy numbers to those of β-actin mRNA.