title: Establishment of a multiplex RT-PCR assay for the rapid detection of fish cytokines.
abstract: To monitor the expression of cytokine genes in Japanese pufferfish, a novel platform for quantitative multiplexed analysis was developed. This custom-designed multiplex RT-PCR assay was used to analyze the expression profiles of 19 cytokine genes, including pro-inflammatory (IL-1β, IL-6, IL-17A/F3, IL-18, TNF-α, TNF-N), anti-inflammatory (IL-4/13A, IL-4/13B, IL-10), T-cell proliferation/differentiation (IL-2, IL-15, IL-21, TGF-β1), B-cell activation/differentiation (IL-7, IL-6, IL-4/13A, IL-4/13B), NK cell stimulation (IL-12p35 and IL-12p40), induction of anti-viral activity (I-IFN-1 and IFN-γ), and monocyte/macrophage progenitor cell proliferation (M-CSF1b) cytokines in head kidney cells under immune stimulatory conditions. The expression profiles were dissimilar in the unstimulated control and immune-stimulated cells. Moreover, increased expression profile was observed due to different stimulations for IL-1β, IL-6, IL-10, IL-12p35, IL-12p40, IL-21, TNF-α, TNF-N, I-IFN-1 and IFN-γ genes. These results suggest that cytokine genes could be used as biomarkers to know the immune status of fish. The constructed multiplex RT-PCR assay will enhance understanding on immune regulation by cytokines in fish.
authors.affiliation: Interdisciplinary Research Organization, University of Miyazaki, 1-1 Gakuen kibanadai-nishi, Miyazaki 889-2192, Japan. tkono@cc.miyazaki-u.ac.jp
authors.forename: Tomoya
authors.initials: T
authors.lastname: Kono
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Takayama
authors.forename: Ryusuke
authors.initials: R
authors.lastname: Nagamine
authors.forename: Hiroki
authors.initials: H
authors.lastname: Korenaga
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sakai
mesh_terms: D000818:Animals; D001483:Base Sequence; D016207:Cytokines / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D029941:Fish Proteins / Q000235:genetics*; D020869:Gene Expression Profiling / Q000379:methods / Q000662:veterinary; D007114:Immunization; D007371:Interferon-gamma / Q000235:genetics; D016753:Interleukin-10 / Q000235:genetics; D053583:Interleukin-1beta / Q000235:genetics; D015850:Interleukin-6 / Q000235:genetics; D007668:Kidney / Q000276:immunology; D008213:Lymphocyte Activation / Q000235:genetics; D008214:Lymphocytes / Q000276:immunology; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D023721:Tetraodontiformes / Q000235:genetics* / Q000276:immunology*
chemical_list: D016207:Cytokines; D017931:DNA Primers; D029941:Fish Proteins; D053583:Interleukin-1beta; D015850:Interleukin-6; D016753:Interleukin-10; D012313:RNA; D007371:Interferon-gamma