Department of Environment and Agro-Biotechnologies (EVA), Centre de Recherche Public, Gabriel Lippmann, Belvaux, Luxembourg.
Plant Cell Rep. 2012 Jan;31(1):205-16. doi: 10.1007/s00299-011-1156-1. Epub 2011 Oct 18.
Due to its reproducibility and sensitivity, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) has become the method of choice for quantifying gene expression. However, the accuracy of RT-qPCR is prone to bias if proper precautions are not taken, e.g. starting with intact, non-degraded RNA, considering the PCR efficiency and using the right reference gene(s) for normalization. It has been reported that some of the well-known reference genes are differentially regulated under certain experimental conditions suggesting that there is no gene that could be used as a universal reference. This paper aims at selecting the most suitable reference gene(s) out of six putative genes to be used as normalizer(s) for quantification of gene expression in the grapevine-downy mildew interaction as well as upon induced resistance with chemical elicitors. Moreover, the paper aims at determining the optimal number of reference genes to be used in normalization, since it has been emphasized in the literature that using multiple reference genes increases accuracy. Two different software tools, geNorm and Normfinder, were used to identify the most stable reference genes in grapevine under the aforementioned conditions. The importance of the choice of adequate reference genes is highlighted by studying chitinase expression.
由于其可重复性和灵敏度，实时定量逆转录聚合酶链反应（RT-qPCR）已成为定量基因表达的首选方法。然而，如果不采取适当的预防措施，例如从完整、未降解的 RNA 开始，考虑 PCR 效率并使用正确的参考基因进行归一化，RT-qPCR 的准确性容易出现偏差。据报道，一些知名的参考基因在某些实验条件下存在差异表达，这表明没有一种基因可以作为通用参考。本文旨在从六个假定基因中选择最合适的参考基因（s），用作葡萄霜霉病互作以及化学诱导剂诱导抗性中基因表达定量的归一化因子。此外，本文旨在确定用于归一化的最佳参考基因数量，因为文献强调使用多个参考基因可以提高准确性。使用 geNorm 和 Normfinder 这两种不同的软件工具，确定了在上述条件下葡萄中最稳定的参考基因。通过研究几丁质酶的表达，强调了选择合适参考基因的重要性。
