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

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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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Using FlyAtlas to identify better Drosophila melanogaster models of human disease.

Venkateswara Chintapalli, Jing WANG, Julian Dow … (2007)

FlyAtlas, a new online resource, provides the most comprehensive view yet of expression in multiple tissues of Drosophila melanogaster. Meta-analysis of the data shows that a significant fraction of the genome is expressed with great tissue specificity in the adult, demonstrating the need for the functional genomic community to embrace a wide range of functional phenotypes. Well-known developmental genes are often reused in surprising tissues in the adult, suggesting new functions. The homologs of many human genetic disease loci show selective expression in the Drosophila tissues analogous to the affected human tissues, providing a useful filter for potential candidate genes. Additionally, the contributions of each tissue to the whole-fly array signal can be calculated, demonstrating the limitations of whole-organism approaches to functional genomics and allowing modeling of a simple tissue fractionation procedure that should improve detection of weak or tissue-specific signals.

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Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress.

Nathalie Nicot, Jean-Francois Hausman, Lucien Hoffmann … (2005)

Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. Real-time RT-PCR is at present the most sensitive method for the detection of low abundance mRNA. To avoid bias, real-time RT-PCR is referred to one or several internal control genes, which should not fluctuate during treatments. Here, the non-regulation of seven housekeeping genes (beta-tubulin, cyclophilin, actin, elongation factor 1-alpha (ef1alpha), 18S rRNA, adenine phosphoribosyl transferase (aprt), and cytoplasmic ribosomal protein L2) during biotic (late blight) and abiotic stresses (cold and salt stress) was tested on potato plants using geNorm software. Results from the three experimental conditions indicated that ef1alpha was the most stable among the seven tested. The expression of the other housekeeping genes tested varied upon stress. In parallel, a study of the variability of expression of hsp20.2, shown to be implicated in late blight stress, was realized. The relative quantification of the hsp20.2 gene varied according to the internal control and the number of internal controls used, thus highlighting the importance of the choice of internal controls in such experiments.

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Twenty-five years of quantitative PCR for gene expression analysis.

Heather D VanGuilder, Kent E. Vrana, Willard M. Freeman (2008)

Following its invention 25 years ago, PCR has been adapted for numerous molecular biology applications. Gene expression analysis by reverse-transcription quantitative PCR (RT-qPCR) has been a key enabling technology of the post-genome era. Since the founding of BioTechniques, this journal has been a resource for the improvements in qPCR technology, experimental design, and data analysis. qPCR and, more specifically, real-time qPCR has become a routine and robust approach for measuring the expression of genes of interest, validating microarray experiments, and monitoring biomarkers. The use of real-time qPCR has nearly supplanted other approaches (e.g., Northern blotting, RNase protection assays). This review examines the current state of qPCR for gene expression analysis now that the method has reached a mature stage of development and implementation. Specifically, the different fluorescent reporter technologies of real-time qPCR are discussed as well as the selection of endogenous controls. The conceptual framework for data analysis methods is also presented to demystify these analysis techniques. The future of qPCR remains bright as the technology becomes more rapid, cost-effective, easier to use, and capable of higher throughput.

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Author and article information

Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group

ISSN (Electronic): 2045-2322

Publication date (Electronic): 16 March 2017

Publication date Collection: 2017

Volume: 7

Electronic Location Identifier: 43618

Affiliations

[1 ]School of Biological Sciences, Universiti Sains Malaysia , 11800 Penang, Malaysia

[2 ]Vector Control and Research Unit, School of Biological Sciences, Universiti Sains Malaysia , 11800 Penang, Malaysia

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[a ] ghows@ 123456usm.my

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Publisher Item ID: srep43618

DOI: 10.1038/srep43618

PMC ID: 5353741

PubMed ID: 28300076

SO-VID: e04856e7-be30-412f-a93e-2766994833d7

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

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Zika Virus

Most cited references 52

Using FlyAtlas to identify better Drosophila melanogaster models of human disease.

Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress.

Twenty-five years of quantitative PCR for gene expression analysis.

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