Abi 7500 system platform

Total RNA was extracted from PBMCs treated with the FimA fusion protein with or without transfection with siTLR4 using TRIzol^® reagent (Thermo Fisher Scientific, Inc.). The extracted RNA was then transcribed into double-strand cDNAs using a commercial Reverse Transcription kit (Invitrogen; Thermo Fisher Scientific, Inc.). The cDNA was amplified using the following primers: TLR4 forward, 5′-CCG CTT TCA CTT CCT CTC AC-3′; TLR4 reverse, 5′-CAT CCT GGC ATC ATC CTC AC-3′; nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) forward, 5′-CTT GCT TAG TTG GTC CTC-3′; NF-κB reverse, 5′-ACC CGA AGA GAA ACG A-3′; myeloid differentiation primary response 88 (MyD88) forward, 5′-AGA TGG ACC TCG GGA G-3′; MyD88 reverse, 5′-ATC AAT CAC GCA CGA TTT-3′; β-actin forward, 5′-TCC CTG TAT GCC TCT G-3′; β-actin reverse, 5′-ATG TCA CGC ACG ATT-3′. The reaction system contained cDNA template (1 μl), primers (1 μl), 2X SYBR Green Mix (10 μl; Shanghai GeneCore BioTechnologies Co., Ltd., Shanghai, China) and RNase-free water (8 μl), with a total volume of 20 μl. PCR was performed using the ABI 7500 system platform (Applied Biosystems; Thermo Fischer Scientific, Inc.). The 2^−ΔΔCq method was used to calculate the relative gene expression of TLR4, NF-κB and MyD88 normalized to β-actin (20 (link)). PCR amplification was repeated in triplicate for each gene.