Ma Kang-Sheng, Li Fen, Liang Ping-Zhuo, Chen Xue-Wei, Liu Ying, Gao Xi-Wu
Department of Entomology, China Agricultural University, Beijing 100193, People's Republic of China (
J Insect Sci. 2016 Feb 5;16(1). doi: 10.1093/jisesa/iew003. Print 2016.
To obtain accurate and reliable results from quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis, it is necessary to select suitable reference genes as standards for normalizing target gene expression data. QRT-PCR is a popular analytical methodology for studying gene expression and it has been used widely in studies of Aphis gossypii Glover in recent years. However, there is absence of study on the stability of the expression of reference genes in A. gossypii. In this study, eight commonly used candidate reference genes, including 18S, 28S, β-ACT, GAPDH, EF1α, RPL7, α-TUB, and TBP, were evaluated under various experimental conditions to assess their suitability for use in the normalization of qRT-PCR data. The optimal number of reference genes was determined using the geNorm program, and the suitability of particular reference genes was empirically validated by performing normalizations of expression data for the HSP70 gene. The results showed the most suitable combinations of reference genes for the different experimental conditions. For experiments based on divergent developmental stages, EF1α, β-ACT, and RPL7 are the optimal reference gene combination, both EF1α and β-ACT are the optimal combination used in the experiments of different geographical populations, whereas for experiments of the temperature changes, the combination of GAPDH and RPL7 is optimal, both 18S and β-ACT are an optimal combination for feeding assay experiments. These research results should be useful for the selection of the suitable reference genes to obtain reliable qRT-PCR data in the gene expression study of A. gossypii.
为了从定量实时逆转录聚合酶链反应（qRT-PCR）分析中获得准确可靠的结果，有必要选择合适的内参基因作为标准化靶基因表达数据的标准。qRT-PCR是一种用于研究基因表达的常用分析方法，近年来已广泛应用于棉蚜的研究中。然而，目前尚无关于棉蚜内参基因表达稳定性的研究。在本研究中，评估了8个常用的候选内参基因，包括18S、28S、β-肌动蛋白（β-ACT）、甘油醛-3-磷酸脱氢酶（GAPDH）、延伸因子1α（EF1α）、核糖体蛋白L7（RPL7）、α-微管蛋白（α-TUB）和TATA盒结合蛋白（TBP）在各种实验条件下的情况，以评估它们用于qRT-PCR数据标准化的适用性。使用geNorm程序确定最佳内参基因数量，并通过对热休克蛋白70（HSP70）基因的表达数据进行标准化，从经验上验证特定内参基因的适用性。结果显示了不同实验条件下最合适的内参基因组合。对于基于不同发育阶段的实验，EF1α、β-ACT和RPL7是最佳内参基因组合；EF1α和β-ACT都是不同地理种群实验中使用的最佳组合；而对于温度变化实验，GAPDH和RPL7的组合是最佳的；18S和β-ACT都是取食试验实验的最佳组合。这些研究结果对于在棉蚜基因表达研究中选择合适的内参基因以获得可靠的qRT-PCR数据应该是有用的。
