Real-Time Quantitative PCR (RT-qPCR)

Total RNA in the ileal tissue was extracted by Trizol^® Plus RNA Purification Kit (Thermo Fisher) in accordance with the manufacturer’s instructions. Briefly, 50-100 mg tissue samples were ground into a powder with a tissue homogenizer, and then transferred to a tube with 1 ml of Trizol and incubated at room temperature for 5 mins. Added 0.2 ml chloroform and shook the tube vigorously by hand for 15 s, then incubated at room temperature for 2-3 mins. After being centrifuged at 12000 × g for 15 mins at 4°C, the supernatant was transferred to a new tube, then RNA was precipitated with an equal volume of 70% ethanol. The quantity and quality of RNA were evaluated by a spectrophotometer (NanoDrop-2000, Thermo Fisher Scientific, MA, USA). cDNA was synthesized by SuperScript™ III First-Strand Synthesis SuperMix (Thermo Fisher). RT-qPCR was performed on an ABI prism 7700 Sequence Detector System (Applied Biosystems, Foster City, CA, USA). The reaction scheme was as follows: pre-incubation at 95°C for 1 min, then performing 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 25 s. Then, a melting curve analysis was conducted to affirm the specificity and reliability of the PCR products. Using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal reference gene, the relative expression of mRNA was calculated by the2^−ΔΔCt method (30). The primers designed in this study are listed in Table 1 .

Primers for gene expression analysis using RT-Qpcr.