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Journal of Animal and Veterinary Advances

Year: 2011  |  Volume: 10  |  Issue: 24  |  Page No.: 3254 - 3262

Lipopolysaccharide Stimulus Induces Gene Expression of Cytokines andToll-Like Receptor 2/4 in Ovine Primary Alveolar Macrophages

Jinliang Sheng, Chuangfu Chen, Xia Yang, Yuanzhi Wang, Pengyan Wang and Hui Zhang

Abstract

In this study, Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR) assays were performed with EvaGreen to investigate the dynamics of cytokine (Interleukin (IL)-1β, IL-8, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Interferon (IFN) (γ) and Toll-Like Receptor 2/4 (TLR2/4) gene expression in ovine primary Alveolar Macrophages (AMs) following Lipopolysaccharide (LPS) stimulation. Expression of cytokine and TLR2/4 mRNA was quantified by comparison of Cycle threshold (CT) values with a standard curve generated from plasmid DNA containing the target gene. Examination of LPS-stimulated ovine AMs revealed that cytokine mRNA expression peaked between 4 and 12 h with the exception of IFN-γ mRNA which peaked around 16 h post stimulation. Furthermore, TLR2 and TLR4 mRNA expression rapidly increased post-stimulation and peaked 20 min post-stimulation at a level which was maintained throughout the procedure. In summary, a sensitive and reliable real-time RT-PCR protocol was implemented for the analysis of ovine TLR2/4 and cytokine gene expression profiles.

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