Identification of Reference Genes for Normalizing RNA Expression in Potato Roots Infected with Cyst Nematodes

Abstract

Potato cyst nematodes induce changes in plant host gene expression following root invasion. For accurate comparison of gene expression by reverse transcription quantitative real-time PCR (RT-qPCR), internal reference genes are necessary for transcript normalization. Very few experimental data on suitable reference genes are available for interactions between plant and root pathogens. In this study, we tested eight potential candidate reference genes, for normalizing levels of potato gene transcripts by RT-qPCR, after inoculation by nematodes. Ranking of candidate reference genes was performed using RefFinder WEB-based software. Four reference genes, RPN7 (26S proteasome regulatory subunit), UBP22 (Ubiquitin-specific protease 22), OXA1 (OXA1 protein) and MST2 (mercaptopyruvate sulfurtransferase), were stably expressed in roots of susceptible or resistant potato plants, infected or uninfected by Globodera pallida. A normalization factor based on data from these four genes, highly homologous between potato and tomato, was used to normalize the expression of a chitinase gene, which was induced by nematodes in roots of potatoes carrying the resistance allele at a low effect QTL, GpaXI spl .

Reference Gene Validation for Quantitative RT-PCR during Biotic and Abiotic Stresses in Vitis vinifera

“…Though all aim to determine which candidate genes are the most stable under certain conditions, they run under different algorithms and mathematical models. Therefore, the stability ranking of the putative reference genes might differ depending on the program used [46] . GeNorm analysis relies on the intuitive principle that the expression ratio of two ideal reference genes should always remain constant across all samples.…”

Selection of suitable reference genes for reverse transcription quantitative real-time PCR studies on different experimental systems from carrot (Daucus carota L.)

“…Due to the variability among softwares, RefFinder comprehensive ranking can represent a strategy on the selection of candidate genes. It was previously used to rank candidate reference genes in several other experimental systems in different plant species (Castro-Quezada et al, 2013;Figueiredo et al, 2013;Ovesna et al, 2012;Yeap et al, 2014;Zhu et al, 2012).…”

Identification and validation of reference genes for real-time qPCR normalization during Al-induced programmed cell death in peanut

“…For more reliable and consistent results, the RefFinder was applied to calculate the geometric mean of each candidate gene and to re-rank the stability (Castro-Quezada et al 2013, Zsóri et al 2013. To execute, the ranking of each gene by geNorm, NormFinder and BestKeeper was multiplied by different weighting coefficients and calculated the geometric mean to produce an overall final ranking (Freitas et al 2017).…”