Department of Biotechnology, Ghent University, Ghent, Belgium.
Department of Data Analysis and Mathematical Modelling, Ghent University, Ghent, Belgium.
Planta. 2019 Dec;250(6):2101-2110. doi: 10.1007/s00425-019-03289-x. Epub 2019 Oct 3.
MicroRNAs miR390-5p, miR7694-3p miR1868 and miR1849 were found to be suitable miRNA reference genes for rice, under either infection with the root-knot nematode Meloidogyne graminicola or treatment with BABA. RT-qPCR is a widely used method to investigate the expression levels of genes under certain conditions. A key step, however, to have reliable results is the normalization of expression. For every experimental condition, suitable reference genes must be chosen. These reference genes must not be affected by differences in experimental conditions. MicroRNAs are regulatory RNA molecules, able to direct the expression levels of protein coding genes. In plants, their attributed functions range from roles in development to immunity. In this work, microRNAs (miRNAs) are evaluated for their suitability as reference genes in rice after infection with root-knot nematode Meloidogyne graminicola or after priming with beta-amino butyric acid. The evaluation was based on their amplification efficiency and their stability estimates according to geNorm, NormFinder and BestKeeper. All tested miRNAs, excluding one, were considered acceptable for normalization. Furthermore, miRNAs were validated using miRNA sequencing data. The set of microRNAs miR390-5p and miR7694-3p was found to be the most stable combination under the tested conditions. Another miRNA set consisting of miR7694-3p, miR1868 and miR1849 also shows potential to be used for miRNA expression normalization under experimental conditions beyond the scope of this study. This work is the first report on reference miRNAs in rice for the purpose of plant defence studies.
MicroRNAs miR390-5p、miR7694-3p、miR1868 和 miR1849 被发现是适合水稻的 miRNA 参考基因，无论是在根结线虫 Meloidogyne graminicola 感染还是 BABA 处理下。RT-qPCR 是一种广泛用于研究特定条件下基因表达水平的方法。然而，要获得可靠的结果，关键步骤是进行表达的归一化。对于每个实验条件，都必须选择合适的参考基因。这些参考基因不应受实验条件差异的影响。MicroRNAs 是调节 RNA 分子，能够指导蛋白质编码基因的表达水平。在植物中，它们的功能范围从发育到免疫。在这项工作中，microRNAs (miRNAs) 被评估为在感染根结线虫 Meloidogyne graminicola 或用β-氨基丁酸引发后在水稻中作为参考基因的适用性。评估基于它们的扩增效率和根据 geNorm、NormFinder 和 BestKeeper 估计的稳定性。除了一个之外，所有测试的 miRNAs 都被认为适合归一化。此外，还使用 miRNA 测序数据对 miRNAs 进行了验证。在所测试的条件下，miR390-5p 和 miR7694-3p 的 miRNA 组合被认为是最稳定的。另一个由 miR7694-3p、miR1868 和 miR1849 组成的 miRNA 集也显示出在本研究范围之外的实验条件下用于 miRNA 表达归一化的潜力。这项工作是首次报道用于植物防御研究的水稻参考 miRNA。
