Cytokine expression in respiratory syncytial virus-infected mice as measured by quantitative reverse-transcriptase PCR
Cytokine expression in respiratory syncytial virus-infected mice as measured by quantitative reverse-transcriptase PCR
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DOI：
10.1016/s0166-0934(02)00211-2
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发表时间：
2003-02-01
影响因子：
3.1
通讯作者：
Tang, YW
中科院分区：
文献类型：
--
作者：
Deng, XQ;Li, HJ;Tang, YW
在呼吸道合胞病毒（RSV）感染的小鼠模型中，接种灭活（即福尔马林灭活、明矾沉淀）病毒（KV）或活病毒（LV）疫苗诱导的细胞因子模式已被证明会影响疾病的表达。为了确定用 RSV 攻击的 BALB/c 小鼠中细胞因子 IL-4 和 IFN-7 的 mRNA 表达，开发了实时定量逆转录酶 PCR 测定。该测定使用 5'-核酸外切酶荧光探针，并在 ABI PRISM 7700 序列检测器系统 (TaqMan) 上进行。将 IL-4 和 IFN-7 mRNA 的相对定量水平与分别用于测量 mRNA 和蛋白质水平的 RNase 保护测定 (RPA) 和酶免疫测定 (EIA) 测量的结果进行比较。 TaqMan 测定获得的结果表明，用 KV 引发的小鼠诱导 IL-4 mRNA 产生增加，而 LV 诱导 IFN-γ mRNA 增加，这与传统方法一致。从 TaqMan 获得的 IL-4 和 IFN-γ 相对量与 RPA（IFN-γ 的 r = 0.96，P < 0.01）和 EIA（IL-4 的 r = 0.90，IFN-γ 的 r = 0.75，P < 0.01）测定的相对量高度相关。通过在三次实验中一式三份地测试相同的样品来检查测定的再现性。在测定内和测定间均观察到最小偏差值。 TaqMan 快速、灵敏且可重复，为 RSV 免疫发病机制小鼠模型中细胞因子 mRNA 表达的定量分析提供了另一种工具。 (C) 2002 Elsevier Science B.V. 保留所有权利。
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-7 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-7 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-γ, 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. (C) 2002 Elsevier Science B.V. All rights reserved.