title: 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.
authors: Department of Molecular Biology, Crop Research Institute, v.v.i., Drnovská 507, Ruzyne, 161 06, Prague 6, Czech Republic, janska@vurv.cz.
authors: Anna
authors: A
authors: Janská
authors: Jan
authors: J
authors: Hodek
authors: Pavel
authors: P
authors: Svoboda
authors: Jiří
authors: J
authors: Zámečník
authors: Ilja Tom
authors: IT
authors: Prášil
authors: Eva
authors: E
authors: Vlasáková
authors: Luigi
authors: L
authors: Milella
authors: Jaroslava
authors: J
authors: Ovesná
mesh_terms: D003080:Cold Temperature; D055864:Droughts; D015870:Gene Expression; D018506:Gene Expression Regulation, Plant; D001467:Hordeum / Q000235:genetics* / Q000502:physiology; D018515:Plant Leaves / Q000235:genetics / Q000502:physiology; D010940:Plant Proteins / Q000235:genetics*; D018749:RNA, Plant / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D036226:Seedlings / Q000235:genetics / Q000502:physiology; D013312:Stress, Physiological
chemical_list: D010940:Plant Proteins; D018749:RNA, Plant
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