Real-time PCR

The mice were sacrificed on day 2 after TNBS or oxazolone treatment. We harvested a piece of distal colon approximately 1.0 cm in length from the same segment in all the mice. Colonic mucosa was isolated by careful scraping and RNA was isolated from the colonic mucosa with Trizol (Invitrogen). First-strand cDNAs were synthesized from 2 μg of total RNA in a 20-μl reaction system with M-MLV reverse transcriptase (Invitrogen) and random primers. Real-time PCR was performed using a Bio-RAD IQ5 real-time system and SYBR green PCR Master Mix (Takara Biotechnology Co., Japan). The relative transcription levels of the mRNAs were calculated according to the 2^−ΔΔCt formula. β-2 microglobulin (B2M) was used as an internal control. Mouse real-time PCR primers are shown in Table 1.