Revertra ace qpcr rt kit fsq 101

Total RNA was extracted from bronchoalveolar macrophages with a MiniBEST universal RNA Extraction Kit (9767, Takara, Shiga, Japan) according to the manufacturer’s instructions. The mRNA expression levels of TNF-α, iNOS, IL-10, and Arg-1 were measured by RT-PCR performed using a RT-PCR kit (ReverTra Ace qPCR RT Kit FSQ-101, Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Gene-specific primers were designed to amplify these markers genes. The reference gene β-actin was used as an endogenous control. The primer sequences were as follows: TNF-α Forward: 5′CCCAGACCCTCACACTCAGATCAT-3′, Reverse: 5′-GCAGCCTTGTCCCTTGAAG-AGAA-3′; IL-10 Forward: 5′-CTTTCACTTGCCCTCATCC-3′, Reverse: 5′-ACAAAC-AATACGCCATTCCC-3′; iNOs Forward: 5′-ACATTCAGATCCCGAAACGC-3′, Reverse: 5′-ACAATCCACAACTCGCTCC-3′; Arg-1 Forward: 5′-GGGGAAAGCCAA-GAAG3′, Reverse: 5′TGGTTGTCAGGGGAGTGT-3′; β-actin Forward: 5′-TGGA-ATCCTGTGGCATCCATGAAAC-3′, Reverse: 5′-TAAAACGCAGCTCAGTAACAGT-CCG -3′. The relative mRNA expression levels of each gene was calculated and analyzed with Image J software (NIH, Bethesda, MD, USA).

Dulbecco's modified Eagle's medium/F12 culture medium, fetal bovine serum, penicillin-streptomycin, and 0.25% trypsin were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). penicillin-streptomycin was purchased from CellGro (Corning Incorporated, Corning, NY, USA). Type II collagenase was purchased from Sigma-Aldirch (Merck KGaA, Darmstadt, Germany). Lipofectamine 2000 was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). X-tremeGENE siRNA transfection reagent was purchased from Roche Diagnostics (Indianapolis, IN, USA). ReverTra Ace qPCR RT kit (FSQ-101) and SYBR dye were purchased from Toyobo Life Science (Osaka, Japan). micrON™ agomir-9, micrON™ agomir-control and miR-9 nucleotide fragment were designed and synthesized by Ribo Life Science Co., Ltd. (Soochow, China). Rabbit anti-MMP-13 antibody (sc-30073), mouse anti-collagen type II α1 chain (COL2A1) antibody (sc-52658) and rabbit anti-COL2A1 (sc-28887) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). The dual-luciferase reporter assay system and pGL3-promoter plasmids were purchased from Promega Corporation (Madison, WI, USA).