School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia.
Vector Control and Research Unit, School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia.
Sci Rep. 2017 Mar 16;7:43618. doi: 10.1038/srep43618.
The mosquito Aedes aegypti (Ae. aegypti) is the most notorious vector of illness-causing viruses such as Dengue, Chikugunya, and Zika. Although numerous genetic expression studies utilizing quantitative real-time PCR (qPCR) have been conducted with regards to Ae. aegypti, a panel of genes to be used suitably as references for the purpose of expression-level normalization within this epidemiologically important insect is presently lacking. Here, the usability of seven widely-utilized reference genes i.e. actin (ACT), eukaryotic elongation factor 1 alpha (eEF1α), alpha tubulin (α-tubulin), ribosomal proteins L8, L32 and S17 (RPL8, RPL32 and RPS17), and glyceraldeyde 3-phosphate dehydrogenase (GAPDH) were investigated. Expression patterns of the reference genes were observed in sixteen pre-determined developmental stages and in cell culture. Gene stability was inferred from qPCR data through three freely available algorithms i.e. BestKeeper, geNorm, and NormFinder. The consensus rankings generated from stability values provided by these programs suggest a combination of at least two genes for normalization. ACT and RPS17 are the most dependably expressed reference genes and therefore, we propose an ACT/RPS17 combination for normalization in all Ae. aegypti derived samples. GAPDH performed least desirably, and is thus not a recommended reference gene. This study emphasizes the importance of validating reference genes in Ae. aegypti for qPCR based research.
埃及伊蚊(Aedes aegypti)是传播登革热、基孔肯雅热和寨卡病毒等致病病毒的最臭名昭著的媒介。尽管已经进行了许多利用定量实时 PCR(qPCR)的埃及伊蚊基因表达研究,但目前缺乏一组适合作为该具有流行病学重要性昆虫表达水平标准化参考的基因。在这里,研究了七种广泛使用的参考基因的可用性,即肌动蛋白(ACT)、真核延伸因子 1α(eEF1α)、α-微管蛋白(α-tubulin)、核糖体蛋白 L8、L32 和 S17(RPL8、RPL32 和 RPS17)以及甘油醛 3-磷酸脱氢酶(GAPDH)。在十六个预定的发育阶段和细胞培养中观察到了参考基因的表达模式。通过三个免费可用的算法,即 BestKeeper、geNorm 和 NormFinder,从 qPCR 数据推断出基因的稳定性。这些程序提供的稳定性值生成的共识排名表明,至少需要两个基因进行归一化。ACT 和 RPS17 是最可靠表达的参考基因,因此,我们建议在所有源自埃及伊蚊的样本中使用 ACT/RPS17 组合进行归一化。GAPDH 表现最差,因此不是推荐的参考基因。本研究强调了在基于 qPCR 的埃及伊蚊研究中验证参考基因的重要性。