Dudziak Karolina, Sozoniuk Magdalena, Szczerba Hubert, Kuzdraliński Adam, Kowalczyk Krzysztof, Börner Andreas, Nowak Michał
1Institute of Plant Genetics, Breeding and Biotechnology, University of Life Sciences in Lublin, Akademicka 15, 20-950 Lublin, Poland.
2Chair and Department of Biochemistry and Molecular Biology, Medical University of Lublin, Chodźki 1, 20-093 Lublin, Poland.
Plant Methods. 2020 Apr 25;16:58. doi: 10.1186/s13007-020-00601-9. eCollection 2020.
Quantitative PCR (qPCR) is one of the most common and accurate methods of gene expression analysis. However, the biggest challenge for this kind of examinations is normalization of the results, which requires the application of dependable internal controls. The selection of appropriate reference genes (RGs) is one of the most crucial points in qPCR data analysis and for correct assessment of gene expression. Because of the fact that many reports indicate that the expression profiles of typically used RGs can be unstable in certain experimental conditions, species or tissues, reference genes with stable expression levels should be selected individually for each experiment. In this study, we analysed a set of ten candidate RGs for wheat seedlings under short-term drought stress. Our tests included five 'traditional' RGs (GAPDH, ACT, UBI, TUB, and TEF1) and five novel genes developed by the RefGenes tool from the Genevestigator database.
Expression stability was assessed using five different algorithms: geNorm, NormFinder, BestKeeper, RefFinder and the delta Ct method. In the final ranking, we identified three genes: CJ705892, ACT, and UBI, as the best candidates for housekeeping genes. However, our data indicated a slight variation between the different algorithms that were used. We revealed that the novel gene CJ705892, obtained by means of in silico analysis, showed the most stable expression in the experimental tissue and condition.
Our results support the statement, that novel genes selected for certain experimental conditions have a more stable level of expression in comparison to routinely applied RGs, like genes encoding actin, tubulin or GAPDH. Selected CJ705892 gene can be used as a housekeeping gene in the expression analysis in wheat seedlings under short-term drought. The results of our study will be useful for subsequent analyses of gene expression in wheat tissues subjected to drought.
定量聚合酶链反应（qPCR）是基因表达分析中最常用且准确的方法之一。然而，此类检测面临的最大挑战是结果的标准化，这需要应用可靠的内参。选择合适的内参基因（RGs）是qPCR数据分析及正确评估基因表达的关键要点之一。鉴于许多报告表明，常用内参基因的表达谱在某些实验条件、物种或组织中可能不稳定，因此每个实验都应单独选择表达水平稳定的内参基因。在本研究中，我们分析了一组在短期干旱胁迫下的小麦幼苗的十个候选内参基因。我们的测试包括五个“传统”内参基因（GAPDH、ACT、UBI、TUB和TEF1）以及通过Genevestigator数据库的RefGenes工具开发的五个新基因。
使用五种不同算法评估表达稳定性：geNorm、NormFinder、BestKeeper、RefFinder和ΔCt法。在最终排名中，我们确定了三个基因：CJ705892、ACT和UBI，作为看家基因的最佳候选者。然而，我们的数据表明所使用的不同算法之间存在细微差异。我们发现，通过计算机分析获得的新基因CJ705892在实验组织和条件下表现出最稳定的表达。
我们的结果支持以下观点，即针对特定实验条件选择的新基因与常规应用的内参基因（如编码肌动蛋白、微管蛋白或GAPDH的基因）相比，具有更稳定的表达水平。所选的CJ705892基因可作为短期干旱下小麦幼苗表达分析中的看家基因。我们的研究结果将有助于后续对遭受干旱的小麦组织中的基因表达进行分析。
