Colonic Mucosal RNA Extraction and qPCR

Total RNA from the colonic mucosa of mice was extracted using TRIzol reagent (Invitrogen, USA). First-strand cDNA templates were synthesized using AMV reverse transcriptase (Promega, USA)^{38 (link)} with hexanucleotide random primers. Conventional PCR was carried out in a BioRad thermocycler. Quantitative real-time PCR amplification was carried out using specific primers designed based on cDNA sequences deposited in the GenBank database^{39 (link)} (Table 1). Real-time PCR was performed in a Bio-rad CFX-96 Real-Time PCR system using SYBR green PCR reagent kits (Bio-rad). Briefly, each of the 40 cycles consisted of denaturation at 95 °C for 10 s, primer annealing at 55 °C for 20 s, and extension at 72 °C for 30 s after an initial hot start at 95 °C for 30 s, with a final incubation at 72 °C for 10 min. The relative amount of transcripts was calculated using the 2^−ΔΔCt formula as the described^{40 (link)}, where DCt is the value from the threshold cycle (Ct) value of the treated sample subtracting the Ct value of the untreated or zero time-point control sample. The relative amount of the sample mRNA was normalized to the GAPDH mRNA.