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To normalize the using of quantitative real-time reverse transcription PCR |
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Abstract Quantitative real-time reverse transcription-PCR (RT-qPCR ) is an efficient tool to measure absolute transcript abundance and provides valuable quantitative information on gene expression of biologic samples from different sources. Thousands of research laboratories worldwide have embraced RT-qPCR as a frequently used method for measuring genes expression in transcript levels because of its relatively low cost, high precision, and high sensitivity, as well as flexibility and scalability. However, despite its popularity, more and more researchers begin to realize that the accuracy of RT-qPCR gene expression analysis depends largely on a proper normalization. However, the simplicity of the technology itself makes it vulnerable for abuse in experiments in which the operator does not perform the required quality control throughout the entire procedure. Here, we review entire RT-qPCR workflow and indicated point by point where and how critical issues can be resolved, such as experiment design, sample and assay quality control, selection of proper reference genes for normalization, data analysis and reporting guidelines. Following the advice in this paper, any user should be able to do (more) successful gene expression profiling using the RT-qPCR technology.
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Received: 01 June 2010
Published: 19 October 2010
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