Qpcr 2 premix sybr

The total RNA was isolated from the intestines using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. First-strand cDNA synthesis from the total RNA template was performed using M-MLV cDNA Synthesis Kit (Enzynomics, Daejeon, Korea). The resulting cDNA was subjected to real-time PCR using qPCR 2 × PreMIX SYBR (Enzynomics, Daejeon, Korea) and a QuantStidio3 (Applied Biosystems, TermoFisher Scientific, MA, USA). The expression level of genes was normalized with the housekeeping gene GAPDH. Use the following primers: murine GAPDH, forward, 5′-AGG TCG GTG TGA ACG GAT TTG-3′, reverse, 5′-AGG TTT GAT TCA GGC AGA TGT T-3′; murine IL-6, forward, 5′-TAG TCC TTC CTA CCC CAA TTT CC-3′, reverse, 5′-TTG GTC CTT AGC CAC TCC TTC-3′; murine IL-13, forward, 5′-CCT GGC TCT TGC TTG CCT T-3′, reverse, 5′-GGT CTT GTG TGA TGT TGC TCA-3′; murine IL-10, forward, 5′-ATT TGA ATT CCC TGG GTG AGA AG-3′, reverse, 5′-CAC AGG GGA GAA ATC GAT GAC A-3′; murine ZO-1, forward, 5′-TTT TTG ACA GGG GGA GTG G-3′, reverse, 5′-TGC TGC AGA GGT CAA AGT TCA AG-3′; human IL-8, forward, 5′-TTT TGC CAA GGA GTG CTA AAG A-3′, reverse, R: 5′-AAC CCT CTG CAC CCA GTT TTC-3′.

Total RNA samples were extracted from cells or mouse skin using Tri-RNA Reagent (Favorgen, Ping-Tung, Taiwan). First-strand cDNA synthesis from the total 500 ng of RNA was performed with PrimeScript RT master mix (RR036A, Takara, Shiga, Japan). The thermocycling condition was 15 min at 37 °C and 5 s at 85 °C. Synthesized cDNAs were subjected to real-time PCR with qPCR 2× Premix SYBR (Enzynomics, Korea) using a Stratagene Mx3000p qPCR machine (Agilent Technologies, Santa Clara, CA, USA). The PCR conditions used to amplify all genes were 10 min at 95 °C, 40 cycles of 95 °C for 15 s, and 64 °C for 40 se Expression data were calculated from the cycle threshold (Ct) values using the ΔCt method of quantification. RPLP0 was used for normalization. Oligonucleotides are listed in Supplementary Table S1.