Abi taqman fast universal pcr master mix

Total RNA was isolated from small-intestinal tissue (1.5-cm length of the ileum) using ISOGEN-2 kit (Nippon-gene, Tokyo, Japan). Complementary DNA was acquired using High-Capacity RNA-to-cDNA Kit (ThermoFisher Scientific Inc., Waltham, MA, USA). RT-PCR was performed using Applied Biosystems 7500 Fast RT-PCR system with ABI TaqMan Fast Universal PCR Master Mix (ThermoFisher Scientific Inc.). The thermal cycling method was 50 cycles of amplification at 95°C for 15 s and 60°C for 1 min. The mRNA expression levels for interleukin-1β (IL-1β; encoded by Il1b) and tumor necrosis factor α (TNFα; encoded by Tnfα) were quantified using real-time RT-PCR and standardized to TaqMan glyceraldehyde-3-phosphate dehydrogenase (ThermoFisher Scientific Inc.). The mRNA levels were expressed as ratios of the mean value for vehicle-treated mice. The sequences of PCR primers and TaqMan probes are as follows: for the mouse Il1b, the sense primer was 5′-GCTGCTACTCATTCACTGGCAA-3′, the anti-sense primer was 5′-TGCTGCTGGTGATTCTCTTGTA-3′, and the TaqMan probe was 5′-FAM-ATGATCCCAATGAGTCGGCTGGAGA-TAMRA-3′; for the mouse Tnfα, the sense primer was 5′-TCATGCACCACCATCAAGGA-3′, the anti-sense primer was 5′-GAGGCAACCTGACCACTCTCC-3′, and the TaqMan probe was 5′-FAM-AATGGGCTTTCCGAATTCACTGGAGC-TAMRA-3′.

RNA was isolated using PureLink RNA Mini Kit (Ambion, Thermo Fisher Scientific, Austin, TX, USA) and reverse transcribed to cDNA using RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative real‐time PCR was carried out using ABI TaqMan Fast Universal PCR Master Mix (Thermo Fisher) on Applied Biosystems^® 7500 real‐time PCR System (Applied Biosystems, Waltham, MA, USA). Relative gene expression was normalized to housekeeping gene 18S using ΔΔC_t method. The list of primers can be found in Table S2.