Quantitative Analysis of Jejunal RNA Expression

The total RNA of the jejunal tissues was extracted using the AG RNAex Pro reagent (Accurate Biology, Changsha, China). The purity and concentration of the extracted total RNA were determined using a NanoDrop ND-2000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, US). The total RNA (1 μg) was reversely transcribed into cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Accurate Biology) for quantitative PCR analysis. The real-time quantitative PCR (RT-qPCR) was performed on the Light Cycler^® 480 II Real-Time PCR System (Roche, Basel, Switzerland). Primer sequences were synthesized by the Tsingke Biotechnology Co., Ltd. (Beijing, China) and used in this study (listed in Supplementary Table S1). The RT-qPCR was carried out in a 10-μL reaction system, consisting of 4.2 μl of cDNA template, 0.4 μl of each primer, and 5.0 μl SYBR^® Green mix (AG, Changsha, China). The RT-qPCR conditions were as follows: an initial step at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 5 s and annealing at 60°C for 30 s, and a final extension at 72°C for 30 s. The relative mRNA expression levels were calculated using β-actin as the internal control and the 2^−ΔΔCt method (Rao et al., 2013 (link)).