School of Biological Sciences, Heydon-Laurence building-A08, The University of Sydney, NSW 2006, Australia.
J Insect Physiol. 2011 Jun;57(6):840-50. doi: 10.1016/j.jinsphys.2011.03.014. Epub 2011 Mar 22.
Drosophila melanogaster is one of the most important genetic models and techniques such as reverse transcription quantitative real-time PCR (RT-qPCR) are being employed extensively for deciphering the genetics basis of physiological functions. In RT-qPCR, the expression levels of target genes are estimated on the basis of endogenous controls. The purpose of these reference genes is to control for variations in RNA quantity and quality. Although determination of suitable reference genes is essential to RT-qPCR studies, reports on the evaluation of reference genes in D. melanogaster studies are lacking. We analyzed the expression levels of seven candidate reference genes (Actin, EF1, Mnf, Rps20, Rpl32, Tubulin and 18S) in flies that were injured, heat-stressed, or fed different diets. Statistical analyses of variation were determined using three established software programs for reference gene selection, geNorm, NormFinder and BestKeeper. Best-ranked references genes differed across the treatments. Normalization candidacy of the selected candidate reference genes was supported by an analysis of gene expression values obtained from microarray datasets available online. The differences between the experimental treatments suggest that assessing the stability of reference gene expression patterns, determining candidates and testing their suitability is required for each experimental investigation.
黑腹果蝇是最重要的遗传模式生物之一，人们广泛采用逆转录定量实时 PCR（RT-qPCR）等技术来揭示生理功能的遗传基础。在 RT-qPCR 中，根据内参基因来估计靶基因的表达水平。这些内参基因的目的是控制 RNA 数量和质量的变化。虽然确定合适的内参基因对于 RT-qPCR 研究至关重要，但在黑腹果蝇研究中关于内参基因评估的报道却很少。我们分析了在受到损伤、热应激或不同饮食处理的果蝇中七种候选内参基因（肌动蛋白、EF1、Mnf、Rps20、Rpl32、微管蛋白和 18S）的表达水平。使用三个已建立的软件程序（geNorm、NormFinder 和 BestKeeper）进行统计分析，以确定基因变异。不同处理下最佳排名的参考基因也不同。从在线提供的微阵列数据集获得的基因表达值分析支持所选候选参考基因的归一化候选资格。实验处理之间的差异表明，对于每个实验研究，都需要评估参考基因表达模式的稳定性、确定候选基因并测试其适用性。
