Koramutla Murali Krishna, Aminedi Raghavendra, Bhattacharya Ramcharan
ICAR-National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute Campus, New Delhi-110012, India.
Sci Rep. 2016 May 11;6:25883. doi: 10.1038/srep25883.
Mustard aphid, also known as turnip aphid (Lipaphis erysimi) is a major insect pest of rapeseed-mustard group of crops. Tremendous economic significance has led to substantial basic research involving gene-expression studies in this insect species. In qRT-PCR analysis of gene-expression, normalization of data against RNA variation by using appropriate reference gene is fundamental. However, appropriate reference genes are not known in case of L. erysimi. We evaluated 11 candidate reference genes for their expression stability in 21 samples of L. erysimi subjected to various regimes of experimental treatments. Unlike other studies, we validated true effects of the treatments on the samples either by gene-expression study of an associated marker gene or by biochemical tests. In the validated samples, expression stability of the reference genes was analysed by employing four different statistical softwares geNorm, NormFinder, BestKeeper and deltaCt. Drawing consensus on the results from different softwares, we recommend three best reference genes 16S, RPS18 and RPL13 for normalization of qRT-PCR data in L. erysimi. This study provides for the first time a comprehensive list of suitable reference genes for mustard aphid and demonstrates the advantage of using more than one reference gene in combination for certain experimental conditions.
芥菜蚜,也被称为芜菁蚜(Lipaphis erysimi),是油菜-芥菜类作物的主要害虫。其巨大的经济重要性促使人们对该昆虫物种进行了大量涉及基因表达研究的基础研究。在基因表达的qRT-PCR分析中,使用合适的内参基因对RNA变异进行数据归一化是至关重要的。然而,在芥菜蚜中尚不清楚合适的内参基因。我们评估了11个候选内参基因在21个经受不同实验处理方案的芥菜蚜样本中的表达稳定性。与其他研究不同的是,我们通过对一个相关标记基因的基因表达研究或生化测试来验证处理对样本的真实影响。在经过验证的样本中,使用四种不同的统计软件geNorm、NormFinder、BestKeeper和deltaCt分析内参基因的表达稳定性。通过对不同软件结果达成共识,我们推荐三个最佳内参基因16S、RPS18和RPL13用于芥菜蚜qRT-PCR数据的归一化。本研究首次提供了一份适合芥菜蚜的内参基因综合列表,并证明了在某些实验条件下联合使用多个内参基因的优势。