Quantitative PCR Analysis of Gene Expression

IPEC-J2 cells were harvested and the total RNA was extracted using RNAiso Plus (Takara, Dalian, China) according to the manufacturer’s instructions. The quantity and quality of the isolated RNA were determined by absorbance at 260 and 280 nm [69 (link)]. cDNA was synthesized using a Reverse Transcriptase kit (Takara, Dalian, China). Briefly, quantitative PCR was performed by QuanStudio 6 Flex Real-Time PCR detection system (Applied Biosystems, Foster City, CA, USA) with a total of 10 µL of assay solution containing 5 µL SYBR Green mix (Takara), 0.2 µL Rox, 3 µL deionized H₂O, 1 µL cDNA template, and 0.4 µL each of forward and reverse primers (Qingke, Beijing, China). The relative gene expressions compared with the housekeeping gene β-actin were calculated by 2^−CT [70 (link)].