State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People's Republic of China.
Anal Biochem. 2010 Apr 15;399(2):257-61. doi: 10.1016/j.ab.2009.12.008. Epub 2009 Dec 11.
Quantitative real-time polymerase chain reaction (QRT-PCR) has become one of the most widely used methods for gene expression analysis. However, the expression profile of a target gene may be misinterpreted due to unstable expression of the reference genes under different experimental conditions. Thus, a systematic evaluation of these reference genes is necessary before experiments are performed. In this study, 10 putative reference genes were chosen for identifying expression stability using geNorm, NormFinder, and BestKeeper statistical algorithms in 12 different cucumber sample pools, including those from different plant tissues and from plants treated with hormones and abiotic stresses. EF1alpha and UBI-ep exhibited the most stable expression across all of the tested cucumber samples. In different tissues, in addition to expression of EF1alpha and UBI-ep, the expression of TUA was also stable and was considered as an appropriate reference gene. Evaluation of samples treated with different hormones revealed that TUA and UBI-ep were the most stably expressed genes. However, for abiotic stress treatments, only EF1alpha showed a relatively stable expression level. In conclusion, TUA, UBI-ep, and EF1alpha will be particularly helpful for reliable QRT-PCR data normalization in these types of samples. This study also provides guidelines for selecting different reference genes under different conditions.
实时荧光定量聚合酶链反应（QRT-PCR）已成为基因表达分析最广泛使用的方法之一。然而，由于在不同实验条件下参照基因表达不稳定，目标基因的表达谱可能会被误解。因此，在进行实验之前，有必要对这些参照基因进行系统评估。在这项研究中，选择了 10 个假定的参照基因，使用 geNorm、NormFinder 和 BestKeeper 统计算法，在 12 个不同的黄瓜样本池中鉴定其表达稳定性，这些样本池包括来自不同植物组织的样本，以及用激素和非生物胁迫处理的植物样本。EF1alpha 和 UBI-ep 在所有测试的黄瓜样本中表现出最稳定的表达。在不同的组织中，除了 EF1alpha 和 UBI-ep 的表达外，TUA 的表达也很稳定，被认为是一个合适的参照基因。对不同激素处理的样本进行评估表明，TUA 和 UBI-ep 是表达最稳定的基因。然而，对于非生物胁迫处理，只有 EF1alpha 表现出相对稳定的表达水平。总之，TUA、UBI-ep 和 EF1alpha 将特别有助于这些类型的样本中可靠的 QRT-PCR 数据归一化。本研究还为在不同条件下选择不同的参照基因提供了指导。
