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.

Reference gene selection for qPCR in Ammopiptanthus mongolicus under abiotic stresses and expression analysis of seven ROS-scavenging enzyme genes

“…However, normalization is crucial for qPCR studies to control sample differences, and is commonly achieved by including a small number of invariant reference genes (Borges et al 2011;Fernandez et al 2011;Hong et al 2008). An ideal reference gene would have a consistent expression profile across all possible tissues and experimental conditions (Fernandez et al 2011;Guénin et al 2009;Selim et al 2012). However, many studies showed that internal standards, including some housekeeping genes vary considerably in response to experimental condition changes, for example, ACT (Kim et al 2003), 18S and 28S ribosomal subunits (Nicot et al 2005), and UBQ10 (Stür-zenbaum et al 2001).…”

Selection and validation of garlic reference genes for quantitative real-time PCR normalization

“…First, a stable reference gene in one species may not be suitable in other species. For example, GAPDH is expressed stably in grape of different culture conditions but is unstable in wheat (Long et al 2010;Selim et al 2012), and ACT are expressed stably in tomato of different tissues but are unstable in wheat (Long et al 2010;Mascia et al 2010). Second, reference genes display distinct expression profiles under different experimental conditions Xiao et al 2012).…”

Reference Gene Selection and Validation for the Early Responses to Downy Mildew Infection in Susceptible and Resistant Vitis vinifera Cultivars

“…Reference genes must be validated for each experimental condition [24] and the geometrical averaging of multiple internal control genes should be used [25]. For grapevine, validation of reference genes has been reported for berry development [26], abiotic stress [27] and biotic stress [28],[29]. For grapevine-downy mildew pathosystem, reference genes have been validated for susceptible V. vinifera cultivars from 1 to 7 days post- inoculation with P. viticola , being V-type proton ATPase ( VATP16 ), 60 S ribosomal protein L18 ( 60 S ), ubiquitin conjugating enzyme ( UBQ ) and SAND family protein ( SAND ) reported as the most stable [28], [29].…”