Identification and Validation of Reference Genes for the Normalization of Gene Expression Data in qRT-PCR Analysis in Aphis gossypii (Hemiptera: Aphididae).
Identification and Validation of Reference Genes for the Normalization of Gene Expression Data in qRT-PCR Analysis in Aphis gossypii (Hemiptera: Aphididae).
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棉蚜（半翅目：蚜科）qRT-PCR 分析中基因表达数据标准化的参考基因的鉴定和验证
DOI：
10.1093/jisesa/iew003
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Gao XW
中科院分区：
文献类型：
--
作者：
Ma KS;Li F;Liang PZ;Chen XW;Liu Y;Gao XW
为了从实时定量逆转录聚合酶链式反应（qRT-PCR）分析中获得准确可靠的结果，需要选择合适的内参基因作为目标基因表达数据标准化的标准。 QRT-PCR是研究基因表达的流行分析方法，近年来在棉蚜研究中得到广泛应用。但目前尚缺乏内参基因在棉麻中表达稳定性的研究。在本研究中，在不同的实验条件下对八个常用的候选内参基因（包括18S、28S、β-ACT、GAPDH、EF1α、RPL7、α-TUB和TBP）进行了评估，以评估它们在qRT-PCR数据标准化中的适用性。使用 geNorm 程序确定参考基因的最佳数量，并通过对 HSP70 基因的表达数据进行标准化来根据经验验证特定参考基因的适用性。结果显示了针对不同实验条件的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都是不同地理群体实验中使用的最佳组合，而对于温度变化实验，GAPDH和RPL7组合是最佳组合，18S和β-ACT都是摄食试验的最佳组合 实验。这些研究结果对于棉麻基因表达研究中选择合适的内参基因以获得可靠的qRT-PCR数据具有重要意义。
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.
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