Real-Time RT-PCR Protocol for Colonic Mucosa

Total RNA was extracted from colonic mucosa using an RNeasy plus mini kit (Qiagen GmbH, Hilden, Germany) and from RAW264.7 cells using TRIzol Reagent. We purified RNA using the lithium chloride procedure described previously,^{(25 (link))} since DSS is known to interfere with PCR reactions. The cDNA was synthesized using a reverse transcriptase kit (Invitrogen) with an oligo-dT primer. After cDNA synthesis, real-time RT-PCR (Applied Biosystems, Carlsbad, CA) was performed using SYBR Green PCR master mix (Thermo Fisher Scientific, Waltham, MA). The amplification programs were set as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s. The primer sequences are shown in Table 2. All sample mRNA levels were normalized to GAPDH for animal or β-actin for RAW264.7 cells and the relative mRNA levels were calculated.