P033 A multiplex RT-PCR assay for rapid and sensitive detection of cytokines in fish
Introduction A multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay can detect expression of 30 genes simultaneously within a reaction tube, which is advantageous to the conventional analytical methods where only one gene per single reaction is detected. Therefore, to economize the cost, labour, time, sample number and experiment frequency, a novel platform for quantitative multiplexed analysis was developed for the expression study of cytokine genes in Japanese pufferfish, Takifugu rubripes . Methods Using a custom designed multiplex RT-PCR assay and the GenomeLab Genetic Analysis System (GeXPS), expression profiles were analyzed for 19 cytokine genes including pro-inflammatory [Interleukin (IL)-1 β , IL-6, IL-17A/F3, IL-18, Tumor Necrosis Factor (TNF)- α , and TNF-N], anti-inflammatory (IL-4/13A, IL-4/13B, and IL-10), T-cell proliferation/differentiation [IL-2, IL-12p35, IL-12p40, IL-15, IL-21, and Transforming Growth Factor (TGF)- β 1], activation/differentiation of B-cells (IL-7, IL-6, IL-4/13A, and IL-4/13B), induction of anti-viral activity [type I-Interferon (IFN)-1 and IFN- γ ], and proliferation of monocytes/macrophage progenitor cells [M-Colony Stimulating Factor (CSF)-1 β ] in tissues under immune stimulatory conditions. Results We observed that immune stimulants such as LPS and polyI:C altered cytokine gene expression. The expression profiles were different in the un-stimulated control and each immune-stimulated fish. The expression profile following the pathogenic bacterial injection was similar to that for LPS stimulation. Notably, the type I-IFN-1 and IFN- γ genes showed significant up-regulation by polyI:C stimulation but not by stimulation with LPS or bacteria. Conclusion Based on the results, this technique allows rapid and sensitive analysis of cytokine genes transcribed at different conditions to evaluate the immune status of fish. The constructed multiplex RT-PCR assay scopes further understanding of cytokine network in immune regulation in fish.