Cytokine mRNA quantification by real-time PCR
Cytokine mRNA quantification by real-time PCR
复制标题
DOI：
10.1016/s0022-1759(01)00489-6
复制
发表时间：
2002-01-01
影响因子：
2.2
通讯作者：
Goldman, M
中科院分区：
文献类型：
--
作者：
Stordeur, P;Poulin, LF;Goldman, M
实时 PCR 代表了一种准确定量核酸的新方法。这是通过使用荧光探针实现的，荧光探针有两种形式，即水解探针（也称为 TaqMan 探针）和杂交探针。我们决定将此方法应用于细胞因子 mRNA 定量，这导致我们开发了一种方案，该方案提供了一种在 Lightcycler 仪器上开发和快速执行实时 PCR 的简单方法。这是通过使用免费软件实现的，该软件允许选择水解探针和引物。我们首先证明使用水解探针的方法的重现性优于使用杂交探针获得的方法。然后，我们应用该技术来确定植物血凝素 (PHA) 刺激人外周血单核细胞 (PBMC) 时 IL-1ra、IL-1 beta、IL-5、IL-13、TNF-α 和 IFN-gamma 诱导的动力学。最后，该方法还成功地证明了 IFN-α 诱导人单核细胞中 IL-10 mRNA 的积累。 (C) 2002 Elsevier Science B.V. 保留所有权利。
Real-time PCR represents a new methodology that accurately quantifies nucleic acids. This has been made possible by the use of fluorogenic probes, which are presented in two forms, namely hydrolysis probes (also called TaqMan probes) and hybridisation probes. We decided to apply this methodology to cytokine mRNA quantification and this led us to the development of a protocol that provides an easy way to develop and perform rapidly real-time PCR on a Lightcycler instrument. It was made possible by the use of freely available software that permits a choice of both the hydrolysis probe and the primers. We firstly demonstrated that the reproducibility of the method using hydrolysis probes compares favourably with that obtained with hybridisation probes. We then applied this technique to determine the kinetics of IL-1ra, IL-1 beta, IL-5, IL-13, TNF-alpha and IFN-gamma induction upon stimulation of human peripheral blood mononuclear cells (PBMC) by phytohaemagglutinin (PHA). Finally, the method was also used successfully to demonstrate that IFN-alpha induces IL-10 mRNA accumulation in human monocytes. (C) 2002 Elsevier Science B.V. All rights reserved.