Identification of suitable reference genes for real-time RT-PCR normalization in the grapevine-downy mildew pathosystem

Abstract

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.