An overview of real-time quantitative PCR: Applications to quantify cytokine gene expression
An overview of real-time quantitative PCR: Applications to quantify cytokine gene expression
复制标题
DOI：
10.1006/meth.2001.1261
复制
发表时间：
2001-12-01
期刊：
影响因子：
4.8
通讯作者：
Mathieu, C
中科院分区：
文献类型：
--
作者：
Giulietti, A;Overbergh, L;Mathieu, C
细胞因子谱的分析有助于阐明免疫细胞的功能特性，无论是用于研究还是用于临床诊断。实时逆转录聚合酶链反应（RTPCR）正被广泛用于定量来自细胞、体液、组织或组织活检的细胞因子。作为一种非常强大和灵敏的方法，它可以用于定量细胞因子的mRNA表达水平，这些水平在研究的组织中通常非常低。该方法允许在反应的指数期期间直接检测PCR产物，将扩增和检测结合在一个单一步骤中。在这篇综述中，我们讨论了实时RT-PCR的原理，可用的不同方法和化学，资产和一些陷阱。利用TaqMan化学和7700序列检测系统（Applied Biosystems），详细讨论了对大量小鼠和人类细胞因子以及在免疫系统中发挥作用的其他因子的验证。总之，实时RT-PCR技术非常准确和灵敏，允许高通量，并且可以在非常小的样品上进行;因此，它是用于定量免疫细胞或炎症组织中细胞因子谱的首选方法。(C)2001 Elsevier Science（美国）。
The analysis of cytokine profiles helps to clarify functional properties of immune cells, both for research and for clinical diagnosis. The real-time reverse transcription polymerase chain reaction (RTPCR) is becoming widely used to quantify cytokines from cells, body fluids, tissues, or tissue biopsies. Being a very powerful and sensitive method it can be used to quantify mRNA expression levels of cytokines, which are often very low in the tissues under Investigation. The method allows for the direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in one single step. In this review we discuss the principle of real-time RT-PCR, the different methodologies and chemistries available, the assets, and some of the pitfalls. With the TaqMan chemistry and the 7700 Sequence Detection System (Applied Biosystems), validation for a large panel of murine and human cytokines and other factors playing a role in the immune system Is discussed in detail. In summary, the real-time RT-PCR technique is very accurate and sensitive, allows a high throughput, and can be performed on very small samples; therefore it is the method of choice for quantification of cytokine profiles in immune cells or inflamed tissues. (C) 2001 Elsevier Science (USA).