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Suitable reference genes for RT-qPCR analysis in Dichelops melacanthus (Hemiptera: Pentatomidae).
Molecular Biology Reports ( IF 2.8 ) Pub Date : 2020-06-27 , DOI: 10.1007/s11033-020-05550-z
Daniele Heloísa Pinheiro 1, 2 , Raquel Oliveira Moreira 1, 3 , Natália Alves Leite 1, 4 , Ana Carolina Redoan 1 , André da Silva Xavier 1, 5 , Beatriz de Almeida Barros 1 , Newton Portilho Carneiro 1
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The relative quantification of gene expression is mainly realized through reverse transcription-quantitative PCR (RT-qPCR). However, the accuracy of this technique is deeply influenced by the expression stability of the reference genes used for data normalization. Therefore, the selection of suitable reference genes for a given experimental condition is a prerequisite in gene expression studies. Dichelops melacanthus (Hemiptera: Pentatomidae) is an important phloem sap-sucking insect pest of soybean, wheat, and maize in Brazil. Most of the genetic and molecular biology studies require gene expression analysis. Nevertheless, there are no reports about reference genes for RT-qPCR data normalization in D. melacanthus. In this study, we evaluated the expression stability of nine candidate reference genes (nadh, sdhb, gapdh, fau, ef1a, rpl9, ube4a, gus and rps23) in different developmental stages, body parts, sex, starvation-induced stress and dsRNA exposure by RefFinder software that integrates the statistical algorithms geNorm, NormFinder, BestKeeper, and ΔCt method. Our results showed that ef1a and nadh are the most stable reference genes for developmental stages, fau and rps23 for sex, ube4a and rps23 for body parts, rpl9 and fau for starvation stress, and nadh and sdhb for dsRNA exposure treatment. The reference genes selected in this work will be useful for further RT-qPCR analyses on D. melacanthus, facilitating future gene expression studies that can provide a better understanding of the developmental, physiological, and molecular processes of this important insect pest. Moreover, the knowledge gained from these studies can be helpful to design effective and sustainable pest management strategies.



中文翻译:

适用于lac实双足动物(半翅目:昆虫科)中RT-qPCR分析的合适参考基因。

基因表达的相对定量主要通过逆转录定量PCR(RT-qPCR)实现。但是,该技术的准确性受用于数据标准化的参考基因的表达稳定性的影响。因此,在给定的实验条件下选择合适的参考基因是基因表达研究的前提。Dichelops melacanthus(Hemiptera:Pentatomidae)是巴西大豆,小麦和玉米中的一种重要韧皮部吸汁性害虫。大多数遗传和分子生​​物学研究都需要基因表达分析。然而,尚无关于黑腹果蝇RT-qPCR数据标准化参考基因的报道。在这项研究中,我们评估了九个候选参考基因(nadhsdhbgapdhfauef1arpl9ube4agusrps23)在不同发育阶段,身体部位,性别,饥饿诱导的压力和dsRNA暴露下的表达稳定性。集成了统计算法geNorm,NormFinder,BestKeeper和ΔCt方法的RefFinder软件。我们的结果表明,ef1anadh是发育阶段最稳定的参考基因,faurps23是性别,ube4arps23用于身体部位,RPL9FAU为饥饿应力,和NADHSDHB用于dsRNA的曝光处理。在这项工作中选择的参考基因将有助于进一步对黑斑病病菌进行RT-qPCR分析,促进未来的基因表达研究,从而可以更好地了解这种重要害虫的发育,生理和分子过程。此外,从这些研究中获得的知识可能有助于设计有效和可持续的有害生物管理策略。

更新日期:2020-06-28
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