Horticulture Department, University of Georgia, Athens, GA 30602, USA.
Plant Cell Rep. 2011 Dec;30(12):2167-76. doi: 10.1007/s00299-011-1121-z. Epub 2011 Jul 15.
Application of transcriptomics approaches can greatly enhance our understanding of blueberry physiology. The success of transcriptomics approaches is dependent on the extraction of high-quality RNA which is complicated by the abundance of polyphenolics and polysaccharides in blueberry. Additionally, transcriptomics requires the accurate quantification of transcript abundance. Quantitative real-time polymerase chain reaction (qRT-PCR) is a robust method to determine transcript abundance. Normalization of gene expression using stably expressed reference genes is essential in qRT-PCR. An evaluation of the stability of expression of reference genes has not yet been reported in blueberry. The objectives of this study were to develop an effective procedure for extracting RNA from different organs and to evaluate potential reference genes for qRT-PCR analyses in blueberry. RNA of high quality and yield was extracted from eight and six organs of rabbiteye and southern highbush blueberry, respectively, using a modified cetyltrimethyl ammonium bromide-based method. The expression stability of 12 reference genes was evaluated. UBIQUITIN-CONJUGATING ENZYME (UBC28), RNA HELICASE-LIKE (RH8), CLATHRIN ADAPTER COMPLEXES MEDIUM SUBUNIT FAMILY PROTEIN (CACSa), and POLYUBIQUITIN (UBQ3b) were the most stably expressed genes across multiple organs in both blueberry species. Further, the expression stability of the reference genes in the branch abscission zone following treatment with fruit abscission-inducing compounds was analyzed. CACSa, RH8, and UBC28 were the most stably expressed genes in the abscission zone under abscission-inducing conditions. We suggest a preliminary evaluation of UBC28, CACSa, RH8, and UBQ3b to identify the most suitable reference genes for the experimental conditions under consideration in blueberry.
应用转录组学方法可以极大地增强我们对蓝莓生理学的理解。转录组学方法的成功取决于高质量 RNA 的提取，而蓝莓中多酚和多糖的丰富性使这一过程变得复杂。此外，转录组学需要准确量化转录本丰度。实时定量聚合酶链反应 (qRT-PCR) 是确定转录本丰度的一种强大方法。qRT-PCR 中使用稳定表达的参考基因对基因表达进行标准化是必不可少的。在蓝莓中，尚未有关于参考基因表达稳定性的评价报道。本研究的目的是开发一种从不同器官提取 RNA 的有效方法，并评估 qRT-PCR 分析中蓝莓潜在的参考基因。使用改良的基于十六烷基三甲基溴化铵的方法，从兔眼和南高丛蓝莓的 8 个和 6 个器官中分别提取了高质量和高产量的 RNA。评估了 12 个参考基因的表达稳定性。在两种蓝莓中，泛素结合酶 28（UBC28）、RNA 解旋酶样 8（RH8）、网格蛋白衔接复合物中等亚基家族蛋白（CACSa）和多聚泛素（UBQ3b）是多个器官中表达最稳定的基因。此外，还分析了在使用果实脱落诱导化合物处理后，脱落区中参考基因的表达稳定性。在诱导脱落条件下，CACSa、RH8 和 UBC28 是脱落区表达最稳定的基因。我们建议对 UBC28、CACSa、RH8 和 UBQ3b 进行初步评估，以确定在蓝莓考虑的实验条件下最适合的参考基因。
