Quantitative Gene Expression Analysis

Colon, MLN, and spleen samples were isolated, snap frozen in liquid nitrogen, and stored at −80°C till analysis. Total RNA was extracted using ONE STEP RNA Reagent (BIO BASIC Inc., Markham, Ontario, Canada) according to manufacturer's protocol. RNA purity and concentration was assessed by NanoPhotometer® P-Class P330-30 (Implen, Munich, Germany). In order to purify RNA from all polysaccharides, including DSS, an additional purification step using 8 M LiCl was performed [29 (link)], followed by the standard genomic DNA purification step using Deoxyribonuclease I kit (Sigma Aldrich, St Louis, MO, USA). One microgram of RNA was used for cDNA synthesis by High Capacity cDNA kit with RNase Inhibitor (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed on CFX96 system (Bio Rad, Singapore) to assess relative expression of catalase (CAT), glutathione peroxidase 1 (GPx1), superoxide dismutase 1 (SOD1), HIF-1α, IL-1β, IL-2, and IL-6. Gene expression was normalized to HPRT1 gene. Primers list is given in the supplementary data (Table 1). Except for the primers for IL-6 gene published by Jeong et al. [30 (link)], all other primers were custom made using Primer 3 software. Messenger RNA expression was determined using SsoFast EvaGreen Supermix (Bio Rad, Singapore). Data are presented as mean ± s.e.m.