Option monitor 3 real time pcr detection system

Real-time quantitative PCR was performed in an Option Monitor 3 Real-Time PCR Detection System (Bio-Rad) using the SYBR Green Supermix (TaKaRa). Expression levels of β-actin (housekeeper genes), SGLT1, GLUT2, divalent metal transporter 1 (DMT1), TNF-α, IL-6, ZO-1, Occluding and Claudin-1 in the small intestinal were analysed using SYBR Premix Ex Taq II (Tli RNaseH Plus) reagents (TakaRa) and the QuanStudio 6 Flex Real-Time PCR detection system (Applied Biosystems). All primers were commercially synthesised and purified by Sangon Biotech Co. Ltd and are shown in Table 2. The reaction was performed in a volume of 10 μl consisting of 5 μl of SYBR Premix Ex Taq (2×), 1 μl of reverse primers, 1 μl of forward primers, 2 μl of doubled-distilled water and 1 μl of cDNA template. Cycling conditions were as follows: 5°C for 30 s, followed by forty cycles at 95°C for 5 s, 60°C for 34 s, under melt curve conditions at 95°C for 15 s, 60°C for 1 min and then 95°C for 15 s (temperature change velocity 0•5°C/s). The target gene mRNA expression level was calculated using the 2 -ΔΔCt method (17) . Each sample was repeated in triplicate.