Research Institute of Horticulture, Konstytucji 3 Maja 1/3, 96-100, Skierniewice, Poland.
Mol Biol Rep. 2019 Dec;46(6):5723-5733. doi: 10.1007/s11033-019-05006-z. Epub 2019 Jul 31.
This is the first study which describes a unique procedure of isolating of high-quality, intact RNA from strawberry leaves of Xanthomonas fragariae, three most suitable reference genes, crucial for the normalization of RT-qPCR data for this pathogen and accurate expression analysis of target genes. In our study, various mathematic algorithms: NormFinder geNorm, BestKeeper, the delta CT method, RefFinder were adopted for validation of most stable reference genes from nine candidate genes (ffh, glyA, gyrA, gyrB, proC, pykA, recA, rpoB, rpoD). The analyses allowing to select three most suitable pioneer reference genes, gyrB, ffh, and pykA, that we recommend for the normalization of RT-qPCR data and for the study of the expression of target genes in Xf. Moreover, their combination as references allowed for an accurate expression analysis and computation of the fold change of the flhF and iroN2 genes in Xf. These two genes are important for the success of the colonization of plant tissue and pathogenicity and sequences of primers designed to study these genes, are presented.
这是首例描述从草莓黄单胞菌叶片中分离高质量完整 RNA 的独特方法的研究，同时鉴定了三个最适的内参基因，这对于该病原菌 RT-qPCR 数据的归一化和靶基因的准确表达分析至关重要。在本研究中，采用各种数学算法（NormFinder、geNorm、BestKeeper、delta CT 法、RefFinder）从 9 个候选基因（ffh、glyA、gyrA、gyrB、proC、pykA、recA、rpoB、rpoD）中验证最稳定的内参基因。分析结果可选择三个最适的内参基因，gyrB、ffh 和 pykA，我们推荐用于 RT-qPCR 数据的归一化以及 Xf 中靶基因表达的研究。此外，它们的组合作为参照可准确分析和计算 Xf 中 flhF 和 iroN2 基因的表达倍数变化。这两个基因对于成功定殖植物组织和致病性非常重要，并提出了用于研究这些基因的引物序列。
