Quantitative Analysis of Inflammatory Gene Expression

Total RNA was isolated from the colonic tissues with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Total RNA (2 µg) was reverse-transcribed using oligo-dT primer (Applied Biosystems, Branchburg, NJ, USA), and real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed using a 7900H Fast Real-Time PCR System (Applied Biosystems) as described previously [14 (link)]. The set of primers for rat IL-1β, IL-6, IL-18, IFN-γ, TNF-α, ZO-1, occludin, claudins, and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were prepared as shown in Table 1. Real-time PCR assays were performed using 100 ng of RNA equivalent cDNA, SYBR-Green Master Mix (Applied Biosystems) and 500 nmol/L gene specific primers. The PCR cycling conditions were 95 °C for 15 s and 60 °C for 60 s. The intensity of the fluorescent dye was determined, and the expression levels of target genes mRNA were normalized to the expression level of GAPDH mRNA.