Trzol reagent extraction kit

The total RNA was extracted using the TRZol reagent extraction kit (Invitrogen Life Technologies, Inc). The mRNA expressions of TLR4, NF-κB, MIF, MadCAM-1, and GAPDH in each group were detected by real-time PCR. The sequences of the PCR primers are shown in Table 3. Ct of every sample was analyzed by MxPro-Mx3000p software. The relative amount of each gene was calculated by utilizing the expression of GAPDH as an internal control by using equation 2△△Ct where ΔCt = (Ct gene-Ct GAPDH). △△Ct ＝ △Ct － average (△Ct normal), cDNA ＝ 2△△Ct. All real-time PCR experiments were performed with Power SYBR Green PCR Master Mix (Applied Biosystems company, USA).

Total RNA from lungs and bowel were extracted using the TRZol reagent extraction kit (Invitrogen, CA, USA). The mRNA expressions of TLR4, NF-κB, MIF, MadCAM1, and SP-A in the lung and bowel were detected by Realtime-PCR. The sequences of the PCR primers were shown in Table 1. Ct of every sample was analysed by MxPro-Mx3000p software. The relative amount of each gene was calculated by utilizing the expression of GAPDH as internal control and using the equation 2^△△Ct where ΔCt = (Ct gene-Ct GAPDH). △△Ct=△Ct−average (△Ct normal), cDNA=2^△△Ct. All Real-time PCR experiments were performed with Pwer SYBR Green PCR Master Mix (Applied Biosystems company, USA).