College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.
Mol Biotechnol. 2011 Oct;49(2):192-7. doi: 10.1007/s12033-011-9394-6.
Quantitative real-time PCR (RT-qPCR) is a reliable method for assessing gene expression, provided that suitable reference genes are included to normalize the data. The stability of expression of eight potential reference genes, namely, tubulin (alpha-2,4 tubulin), actin, EF1 α (elongation factor 1 α), UBC (ubiquitin C), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), psaA (photosynthesis-related plastid gene representing photosystem I), PP2Acs (catalytic subunit of protein phosphatase 2A), and PGK (phosphoglycerate kinase), was assessed in chrysanthemum plants subjected to aphid infestation, heat stress or waterlogging stress using geNorm software. The widely used reference gene EF1 α performed well for aphid infested plants but poorly for waterlogged ones. The catalytic subunit of protein phosphatase 2A (PP2Acs) was the best performing one during heat and waterlogging stress, but was the worst during aphid infestation. The commonly used reference gene actin was generally the least stable of the set. No single gene was suitable for normalization on its own. The choice of reference gene(s) is an important factor in gene expression studies based on RT-qPCR.
实时荧光定量 PCR（RT-qPCR）是评估基因表达的一种可靠方法，前提是包含合适的内参基因来对数据进行标准化。在对遭受蚜虫侵害、热胁迫或水淹胁迫的菊花植株进行分析时，使用 geNorm 软件评估了 8 个潜在的内参基因（α-2、4 微管蛋白、肌动蛋白、EF1α（延伸因子 1α）、UBC（泛素 C）、GAPDH（甘油醛-3-磷酸脱氢酶）、代表光系统 I 的与光合作用相关的质体基因 psaA、PP2Acs（蛋白磷酸酶 2A 的催化亚基）和 PGK（磷酸甘油酸激酶））的表达稳定性。广泛使用的内参基因 EF1α 在受蚜虫侵害的植物中表现良好，但在水淹植物中表现不佳。蛋白磷酸酶 2A 的催化亚基（PP2Acs）在热胁迫和水淹胁迫期间表现最佳，但在蚜虫侵害期间表现最差。常用的内参基因肌动蛋白通常是最不稳定的基因之一。没有一个基因可以单独作为标准化的最佳选择。参考基因的选择是基于 RT-qPCR 的基因表达研究中的一个重要因素。
