Validation of Candidate Reference Genes for the Accurate Normalization of Real-Time Quantitative RT-PCR Data in Rice During Seed Development
Validation of Candidate Reference Genes for the Accurate Normalization of Real-Time Quantitative RT-PCR Data in Rice During Seed Development
复制标题
验证候选内参基因，以准确标准化水稻种子发育过程中的实时定量 RT-PCR 数据
DOI：
10.1007/s11105-009-0124-1
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发表时间：
2010-03-01
影响因子：
2.1
通讯作者：
Liu, Qiao-Quan
中科院分区：
文献类型：
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作者：
Li, Qian-Feng;Sun, Samuel S. M.;Liu, Qiao-Quan
水稻种子是淀粉和蛋白质的天然储存器官，也是生产有价值蛋白质的理想生物反应器。越来越多的研究集中在水稻上，试图确定其基因的功能，并提高其产量和质量。实时荧光定量RT-PCR技术具有准确、灵敏、重复性好等优点，是目前基因表达分析的最佳选择。然而，正确选择用于标准化的参考基因是可靠结果的关键前提。本研究利用geNorm软件对水稻中常用的9个看家基因的表达稳定性进行了细致的研究。结果表明，在来自两个水稻品种的几乎所有供试样品中，eIF-4a和ACT1是最适合的参考基因，包括不同的时空特异性组织，尤其是在不同发育阶段的种子中。相反，18S和25S rRNAs这两个常见的参考基因的表达最不稳定。此外，为了在水稻种子发育的时空表达分析中获得更可靠的结果，有必要将多个合适的参考基因一起进行归一化处理。当用来量化水稻种子发育过程中几个感兴趣的基因的表达时，验证的参考基因被进一步依赖。
Rice seed, a natural storage organ for starch and protein, is also an ideal bioreactor for the production of valuable proteins. Increasingly, studies focused on rice have tried to determine the functions of its genes and also to improve its yield and quality. Real-time RT-PCR is the best available choice at present for gene expression analysis due to its accuracy, sensitivity, and reproducibility. The right choice of reference genes for normalization, however, is a critical precondition for reliable results. In this study, the expression stabilities of nine commonly used housekeeping genes in rice were carefully assessed using the software geNorm. Our results showed that eIF-4a and ACT1 were the most suitable reference genes among almost all the tested samples from two rice varieties, including different temporal and spatial-specific tissues, especially in seeds at different developmental stages. In contrast, 18S and 25S rRNAs, two common reference genes, were found to have the least stable expression. Moreover, it is necessary to use multiple suitable reference genes together for normalization to get a more reliable result in temporal and spatial expression analysis during rice seed development. The validated reference genes were further relied when used to quantify the expression of several genes of interest during rice seed development.