7500 instrument

Total RNA was isolated from the liver tissues of broiler chickens using RNAiso Plus reagent (Takara Biotechnology Co., Ltd, Dalian, China) following the manufacturer’s instructions. Total RNA concentration was quantified by measuring the absorbance at 260 nm with a NanoDrop ND-100 spectrophotometer (NanoDrop Technologies, Rockland, DE, United State), and the purity was assessed by determining the ratios of optical density (OD) value at 260 and 280 nm. cDNA was reverse transcribed using a commercial cDNA Synthesis Kit (PrimeScript^TM RT Master Mix, Takara) and diluted 20 times with DEPC water before use. Quantitative real-time PCR was performed on an Applied Biosystems 7500 instrument (Foster City, CA, United State) using SYBR Premix EX Taq (Takara, United State). The PCR reaction conditions consisted of denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, annealing at 60°C for 1 min, and extension at 60°C for 20 s. Primer sets used for quantitative RT-PCR analysis are listed in Supplementary Table 1. All gene expressions are calculated as the relative fold changes compared with CON, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference to normalize the expression of target genes. Relative mRNA expression was calculated according to the 2^−ΔΔCT method.