Abi 7500 fast real time quantitative pcr system

RNAiso (Takara, Dalian, China) was used to extract the total RNA from IPEC-J2. Then, the RNA was reversely transcribed into cDNA using a cDNA synthesis kit (Vazyme Biotech Co., Ltd., Nanjing, China). Based on the sequences published in the GenBank database and Primer Premier 5.0 software, the primers (Table S9) were designed and synthesized by Sangon Biotech (Shanghai, China). The reaction was performed using an ABI 7500 Fast Real-Time Quantitative PCR System (Applied Biosystems, Foster City, CA, USA) with a 20 μL mixture containing 2 μL of cDNA template, 10 μL of 2 × AceQ Universal SYBR qPCR Master Mix, 0.4 μL each of forward and reverse primers, and 7.2 μL of ddH₂O. The reaction was conducted at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. GAPDH was chosen as an internal control, and the relative gene expression was calculated using the 2^−∆∆Ct method [43 (link)].

Total RNA was isolated from cells and intestinal samples using the TRIzol reagent (Takara Biotech, Dalian, China) following the manufacturer’s protocol. RT-qPCR amplification was performed using an ABI 7500 Fast Real-Time Quantitative PCR System (Applied Biosystems, Foster City, CA, USA) and relative gene expression was calculated using the 2^−ΔΔCt method (27 (link)). The housekeeping gene GAPDH was used as an internal control for each experiment. The experiments were performed at least three times with data presented as means values ± SD. The primers are shown in Table 4.