Quantifying Gene Expression in Intestinal Mucosa

Total RNA was isolated from the liquid-nitrogen-pulverized jejunal and ileal mucosa samples with the RNAiso Plus reagent (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s protocol. The concentration and quality of the RNA were determined using the NanoDrop One Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR (AG11728, Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China) following the manufacturer’s instructions. The RT-qPCR was then performed on a LightCycler R480II Real-Time PCR Instrument (Roche, Basel, Switzerland) using the SYBR Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China) in accordance with manufacturer’s instructions. The fluorescence PCR program was set as follows: pre-denaturation, 95 °C for 30 s, 1 cycle; PCR amplification, 95 °C for 5 s, 60 °C for 30 s, 40 cycles; melting, 95 °C for 5 s, 60 °C for 1 min, 1 cycle; cooling, 50 °C for 30 s. Primers used in the PCR assay are listed in Table 1. The relative expression of selected genes normalized by β-actin was calculated using the 2^−ΔΔCt method [22 (link)]. The data were expressed as relative values to those for the CON-NBW group.