The choice of reference gene set for assessing gene expression in barley (Hordeum vulgare L.) under low temperature and drought stress

Abstract

Drought and low temperature are the two most significant causes of abiotic stress in agricultural crops and, therefore, they pose considerable challenges in plant science. Hence, it is crucial to study response mechanisms and to select genes for identification signaling pathways that lead from stimulus to response. The assessment of gene expression is often attempted using real-time RT-PCR (qRT-PCR), a technique which requires a careful choice of reference gene(s) for normalization purpose. Here, we report a comparison of 13 potential reference genes for studying gene expression in the leaf and crown of barley seedlings subjected to low temperature or drought stress. All three currently available software packages designed to identify reference genes from qRT-PCR data (GeNorm, NormFinder and BestKeeper) were used to identify informative sets of up to three reference genes. Interestingly, the data obtained from the separate treatment of leaf and crown have led to the recommendations that HSP70 and S-AMD (and possibly HSP90) to be used as the reference genes for low-temperature stressed leaves, HSP90 and EF1α for low-temperature stressed crowns, cyclophilin and ADP-RF (and possibly ACT) for drought-stressed leaves, and EF1α and S-AMD for drought-stressed crowns. Our results have demonstrated that the gene expression can be highly tissue- or organ-specific in barley and have confirmed that reference gene choice is essential in qRT-PCR. The findings can also serve as guidelines for the selection of reference genes under different stress conditions and lay foundation for more accurate and widespread use of qRT-PCR in barley gene analysis.

Reference Genes for RT-qPCR Analysis of Environmentally and Developmentally Regulated Gene Expression in Alfalfa

“…It is also noteworthy that eEF-1α consistently maintained an M value well below the 0.5 geNorm threshold across all treatments and genetic sources and is another candidate to consider in the validation of reference genes in RT-qPCR analysis of gene expression in alfalfa. This is in agreement with previous reports of eEF-1α stable expression in various species tissues and treatments [10] [32] [35] including in low temperature-treated crowns of barley [36]. Figure 1(b) also reveal that even though GAPDH is frequently used as a housekeeping gene in RT-qPCR studies, it seldom met the requirements for robust analysis of gene expression in our study.…”

Selection and validation of reference house­keeping genes in the J774A1 macrophage cell line for quantitative real-time PCR

“…Herein, we suggest that GAPDH can be considered as an internal control for macrophages, based on its stability and lower variance under various experimental conditions, as shown by BestKeeper analysis and the ∆∆ Ct values. This conclusion was also arrived at in lepidopteran insects, barley, and mouse uterus cells (Teng et al, 2012;Janská et al, 2013;Lin et al, 2013).…”

Endophytic colonization of barley (Hordeum vulgare) roots by the nematophagous fungus Pochonia chlamydosporia reveals plant growth promotion and a general defense and stress transcriptomic response

“…Primers used to validate the 7 genes selected on the basis of their involvement in hormone metabolism and stress response (namely AIAC, GA2ox, JIP23, HSP17, HS1, PR1 and PR3) are listed in Table S1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a normalization gene (Janská et al 2013;Kozera and Rapacz 2013). PCR amplifications were carried out in triplicate from three independent biological samples using FastStart Universal Real-Time PCR MasterMixes (Roche Applied Science, Barcelona, Spain) and the ABI Prism 7000 Sequence Detection System from Applied Biosystems (http://www.lifetechnologies.com/es/en/home/ brands/applied-biosystems.html).…”