Identification of suitable grapevine reference genes for qRT-PCR derived from heterologous species

Abstract

Identification and validation of suitable reference genes that exhibit robust transcriptional stability across many sample types is an absolute requirement of all qRT-PCR experiments. Often, however, only small numbers of reference genes, validated across limited sample types, are available for non-model species. This points to a clear need to assess and validate a wider range of potential reference genes than is currently available. We therefore looked to test and validate a large number of potential reference genes across a wide range of tissue types and treatments to determine the applicability of these reference genes for use in grapevine and other non-model plant species. Potential reference genes were selected based on stability of gene transcription in the model plant species Arabidopsis or due to their common use in the grapevine community. The selected reference genes were analyzed across two datasets consisting of a range of either 'Sauvignon blanc' or 'Pinot noir' tissues. A total of 11 potential reference genes were screened across the two datasets. Gene stability was analyzed by GeNorm, a widely used Excel application, or an ANOVA-based method developed in red clover. Both analysis methods showed that all 11 potential reference genes are stably expressed in the datasets tested, but the rankings of gene stability differed based on the datasets and analysis method used. Furthermore, the transcript stability of these genes, initially identified in Arabidopsis and now validated in grapevine, suggests applicability across a wide range of non-model plant species in addition to their utility in grapevine.

Selection and validation of reliable reference genes for gene expression studies from Monilinia vaccinii-corymbosi infected wild blueberry phenotypes

“…As stated by Samarth and Jameson 29 , the selection of appropriate reference genes from field conditions is more complex than controlled environmental studies. Tashiro et al 30 also pointed out the necessity of reference gene validation in studies involving non-model plant species from heterologous plant population. In this study, we assessed seven RGs for their use as internal controls in gene expression studies of the wild blueberry phenotypes upon monilinia blight infection under field conditions.…”

Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

“…However, despite validation, the positive control has no significant influence on the stability evaluation by the calculation software. Thus, many studies [14, 36–39] evaluated suitable reference genes by calculation software without the positive control.…”

A comparison of root and shoot hydraulics, aquaporin expression and leaf gas exchange between two grapevine cultivars reveals differences in hydraulic control mediated by aquaporins

“…Sequences for the gene-specific primers were used from Tashiro et al (2016) (GAPDH) and Shelden (2008) (ELF, PIP1;1, PIP2;2, PIP2;3, TIP1;1) or designed using NCBI Primer-Blast (Ye et al, 2012) for PIP2;1 and TIP2;1 ( Supplementary Table 1). Log2 ratios for the heatmap were calculated between the mean NRQ of the WW controls and the mean NRQ of WD, ABA or REC.…”