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Assessment of Aedes albopictus reference genes for quantitative PCR at different stages of development
PLOS ONE ( IF 2.9 ) Pub Date : 2018-03-19 , DOI: 10.1371/journal.pone.0194664
Najat Dzaki , Ghows Azzam

Members of the Aedes genus of mosquitoes are widely recognized as vectors of viral diseases. Ae.albopictus is its most invasive species, and are known to carry viruses such as Dengue, Chikugunya and Zika. Its emerging importance puts Ae.albopictus on the forefront of genetic interaction and evolution studies. However, a panel of suitable reference genes specific for this insect is as of now undescribed. Nine reference genes, namely ACT, eEF1-γ, eIF2α, PP2A, RPL32, RPS17, PGK1, ILK and STK were evaluated. Expression patterns of the candidate reference genes were observed in a total of seventeen sample types, separated by stage of development and age. Gene stability was inferred from obtained quantification data through three widely cited evaluation algorithms i.e. BestKeeper, geNorm, and NormFinder. No single gene showed a satisfactory degree of stability throughout all developmental stages. Therefore, we propose combinations of PGK and ILK for early embryos; RPL32 and RPS17 for late embryos, all four larval instars, and pupae samples; eEF1-γ with STK for adult males; eEF1-γ with RPS17 for non-blood fed females; and eEF1-γ with eIF2α for both blood-fed females and cell culture. The results from this study should be able to provide a more informed selection of normalizing genes during qPCR in Ae.albopictus.



中文翻译:

评估白纹伊蚊参考基因在不同发育阶段的定量PCR

蚊的伊蚊属成员被广泛认为是病毒性疾病的媒介。albopictus是其最具入侵性的物种,已知携带登革热,Chikugunya和Zika等病毒。它的新兴重要性使Ae成为可能白化病在遗传相互作用和进化研究的最前沿。然而,到目前为止,还没有描述该昆虫特异性的一组合适的参考基因。9个参考基因,即ACTeEF1-γeIF2αPP2ARPL32RPS17PGK1ILKSTK被评估。在总共十七种样品类型中观察到了候选参考基因的表达模式,按发育阶段和年龄进行了分离。通过三个被广泛引用的评估算法,即BestKeeper,geNorm和NormFinder,从获得的定量数据中推断出基因稳定性。在所有发育阶段,没有单个基因显示令人满意的稳定性。因此,我们建议将PGKILK组合用于早期胚胎。RPL32RPS17用于后期胚胎,所有四龄幼虫和p样品;eEF1-γSTK的成年男性;带RPS17的eEF1-γ对于非血液喂养的女性;和eEF1-γeIF2α一起用于有血缘的雌性和细胞培养。这项研究的结果应该能够为Ae的qPCR期间的标准化基因提供更明智的选择。白化病

更新日期:2018-03-20
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