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Evaluation of reference genes at different developmental stages for quantitative real-time PCR in Aedes aegypti.

Abstract

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.

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  • Authors+Show Affiliations

    ,

    School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia.

    ,

    School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia.

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    School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia.

    ,

    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.

    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.

    Source

    Scientific reports 7: 2017 Mar 16 pg 43618

    Pub Type(s)

    Journal Article

    Language

    eng

    PubMed ID

    28300076