Cytokine expression in respiratory syncytial virus-infected mice as measured by quantitative reverse-transcriptase PCR.
In the murine model for respiratory syncytial virus (RSV) infection, cytokine patterns induced by vaccinations with either killed (i.e. formalin-inactivated, alum-precipitated) virus (KV) or live virus (LV) have been shown to influence disease expression. To determine the mRNA expression of the cytokines IL-4 and IFN-gamma in BALB/c mice challenged with RSV, a real-time quantitative reverse-transcriptase PCR assay was developed. This assay uses 5'-exonuclease fluorogenic probes and is performed on the ABI PRISM 7700 Sequence Detector System (TaqMan). The relative quantitative levels of mRNA for IL-4 and IFN-gamma were compared with those measured by an RNase protection assay (RPA) and an enzyme immunoassay (EIA), which are methods used to measure the levels of mRNA and protein, respectively. Results obtained by the TaqMan assay showed that mice primed with KV induces increased IL-4 mRNA production while LV induces increased IFN-gamma mRNA, which is in agreement with conventional methods. IL-4 and IFN-gamma relative quantities obtained from TaqMan were highly correlated to those determined by RPA (r=0.96 for IFN-gamma, P<0.01) and EIA (r=0.90 for IL-4 and r=0.75 for IFN-gamma, P<0.01). Assay reproducibility was examined by testing a same sample in triplicate at three experiments. Minimal deviation values were observed in both intra- and inter-assays. TaqMan, which is rapid, sensitive and reproducible, provides an alternative tool for the quantitative analysis of cytokine mRNA expression in the murine model of RSV immunopathogenesis.
Deng
Xinqing
X
Division of Infectious Diseases, Department of Medicine, Vanderbilt University Hospital, A3310 MCN, Nashville, TN 37232-2605, USA.
Li
Haijing
H
Tang
Yi Wei
YW
D000818:Animals; D004195:Disease Models, Animal; D007371:Interferon-gamma / Q000096:biosynthesis*; D015847:Interleukin-4 / Q000096:biosynthesis*; D008168:Lung / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D012333:RNA, Messenger / Q000096:biosynthesis; D015203:Reproducibility of Results; D018357:Respiratory Syncytial Virus Infections / Q000276:immunology* / Q000517:prevention & control* / Q000821:virology; D022261:Respiratory Syncytial Virus Vaccines / Q000276:immunology*; D012136:Respiratory Syncytial Viruses / Q000276:immunology*; D019914:Taq Polymerase / Q000378:metabolism; D014611:Vaccination
D012333:RNA, Messenger; D022261:Respiratory Syncytial Virus Vaccines; D015847:Interleukin-4; D007371:Interferon-gamma; D019914:Taq Polymerase