Xiao Zheng, Sun Xiaobo, Liu Xiaoqing, Li Chang, He Lisi, Chen Shangping, Su Jiale
Institute of Horticulture, Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Jiangsu Academy of Agricultural Sciences Nanjing, China.
Front Plant Sci. 2016 Oct 18;7:1547. doi: 10.3389/fpls.2016.01547. eCollection 2016.
The quantitative real-time polymerase chain reaction (qRT-PCR) approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constitutes the first systematic evaluation of potential reference genes in G. Don. Eleven candidate reference genes in different tissues and flowers at different developmental stages of were assessed using the following three software packages: GeNorm, NormFinder, and BestKeeper. The results showed that α (elongation factor 1-alpha), (18s ribosomal RNA), and (ribosomal protein L3) were the most stable reference genes in developing rhododendron flowers and, thus, in all of the tested samples, while tublin () was the least stable. (actin), , , and α were found to be the top four choices for different tissues, whereas was not found to favor qRT-PCR normalization in these tissues. Three stable reference genes are recommended for the normalization of qRT-PCR data in . Furthermore, the expression profiles of (phytoene synthase) and (phytoene dehydrogenase) were assessed using α, , , , and their combination as internals. Similar trends were found, but these trends varied when the least stable reference gene was used. The results further prove that it is necessary to validate the stability of reference genes prior to their use for normalization under different experimental conditions. This study provides useful information for reliable qRT-PCR data normalization in gene studies of .
定量实时聚合酶链反应(qRT-PCR)方法已成为分析靶基因表达模式的一种广泛使用的方法。选择最佳的内参基因是qRT-PCR中基因表达准确标准化的前提条件。本研究首次对羊踯躅中潜在的内参基因进行了系统评估。使用以下三个软件包对羊踯躅不同组织和不同发育阶段花朵中的11个候选内参基因进行了评估:GeNorm、NormFinder和BestKeeper。结果表明,α(延伸因子1-α)、(18s核糖体RNA)和(核糖体蛋白L3)是羊踯躅发育花朵中最稳定的内参基因,因此在所有测试样品中也是最稳定的,而微管蛋白()是最不稳定的。(肌动蛋白)、、和α被发现是不同组织的前四个选择,而未发现有利于这些组织中qRT-PCR的标准化。建议使用三个稳定的内参基因对羊踯躅中qRT-PCR数据进行标准化。此外,使用α、、、及其组合作为内参评估了(八氢番茄红素合酶)和(八氢番茄红素脱氢酶)的表达谱。发现了相似的趋势,但当使用最不稳定的内参基因时,这些趋势有所不同。结果进一步证明,在不同实验条件下用于标准化之前,有必要验证内参基因的稳定性。本研究为羊踯躅基因研究中可靠的qRT-PCR数据标准化提供了有用信息。