RNA Isolation and Quantitative RT-PCR

Total RNA was extracted from the colon tissue in each group with Trizol reagent according to the manufacturer’s instructions. After reverse transcription, complementary DNA was used as templates for PCR. Primers for the inflammatory factors and internal reference were as follows: tumor necrosis factor-α (TNF-α): forward, 5′-CATTTCCACG ATTTCCCAGA-3′, reve-rse, 5′-GGAAAGCCCATTTGAGTCCT-3′; interleukin (IL)-1β: forward, 5′-CTCACAAGCAGAGCACAAGC-3′, reverse, 5′-CAGTCCAGCCCATA CTTTAGG-3′; IL-6: forward, 5′-CGGAGAGGAGACTTCACAGAG-3′, reverse, 5′-CATTTCCACGATTTCCCAGA-3′; GAPDH: forward, 5′-GTATGACTCCACTC ACGGCAAA-3′, reverse, 5′-GGTCTCGCTCCTGGAAGATG-3′. The housekeeping gene GAPDH was used as internal control, and the amount of RNA was calculated by the comparative threshold cycle method as recommended by the manufacturer. Quantitative real-time PCR was carried out by ABI 7500 real-time PCR system (Applied Biosystems, Foster, CA, USA).