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

Najat Dzaki,Intan H Ishak,Karima N Ramli,Ghows Azzam,Azali Azlan

doi:10.1038/srep43618

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.

Highlights

The mosquito Aedes aegypti (Ae. aegypti) is the most notorious vector of illness-causing viruses such as Dengue, Chikugunya, and Zika
Purified quantitative real-time polymerase chain reaction (qPCR) products were sequenced to show specificity and accuracy whereby (a) each primer produced a singular sequence output, and (b) the sequence aligns with the cDNA of the expected gene through BLAST
Through the utilization of algorithms conceptualized for reference gene validation, a suitable panel of genes most robust for normalization are identified for each developmental stage and Aag[2] cell culture

Summary

Introduction

The mosquito Aedes aegypti (Ae. aegypti) is the most notorious vector of illness-causing viruses such as Dengue, Chikugunya, and Zika. The past year has seen the mosquito garnering much international attention due to the roles it may have played in the widespread propagation of Zika, historically the first-reported association of the virus to Ae. aegypti dates as far back as 50 years ago[1] This reputation as a host to such organisms has created an Ae. aegypti research base more revolved around the viruses and parasites it carries, rather than the insect itself. Normalization of data against a ‘housekeeping’ or reference gene is critical, as it compensates for differences in starting cDNA quantities amongst samples caused by variations encountered along the RNA extraction and subsequent reverse transcription steps[8,9]. Application of a singular reference gene for all tissue and morphological types of the developing mosquitos an arguably flawed scientific approach This is especially true for Ae. aegypti as in addition to undergoing complete metamorphosis, the insect spends half its life-cycle as an aquatic organism. There is a need for the stability of reference genes at different points of development to be validated to ensure robustness in gene expression normalization where samples are from individuals of a specific developmental stage

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