Selection and validation of reference genes for functional studies in the Calliphoridae family

Cardoso, Gisele Antoniazzi; Matiolli, Cleverson Carlos; Azeredo-Espin, Ana Maria Lima de; Torres, Tatiana Teixeira

doi:10.1093/jis/14.1.2

Cited by 15 publications

(11 citation statements)

References 40 publications

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“…RPL32 should be avoided when evaluating gene expression in samples from pupal tissue; RPS17 in male and blood fed female adult tissue; and GAPDH in samples derived from non-blood fed female adults as well as Aag2 cells. The frequency with which GAPDH appeared at the lower end of consensus rankings was surprisingly high, as the reliability of the gene as a reference in arthropods has been shown time and time again 43 44 45 . Nevertheless, given the high standard deviation and overall instability of gene as displayed by our data, we are comfortable in suggesting caution when utilizing GAPDH in regards to Ae.…”

Section: Discussionmentioning

confidence: 99%

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

Dzaki

Ramli

Azlan

et al. 2017

Sci Rep

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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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Section: Discussionmentioning

confidence: 99%

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

Dzaki

Ramli

Azlan

et al. 2017

Sci Rep

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“…Until now, only a few studies were on the reference evaluation in blowfly species. Cardoso et al 2014 suggested that Actin, GAPDH, and Rpl49 were the most stable genes among life stages in three Calliphoridae species ( Cochliomyia hominivorax Coquerel, C. macellaria Fabricius, and C. albiceps Wiedemann) by designing primers with conserved regions of six candidate reference genes [ 34 ]. In the Australian sheep blowfly, Lucilia cuprina , 18S, 28SrRNA, GST1 (Glutathione-S-transferase), β-TUB and RPLPO (Acidic ribosomal phosphoprotein PO) were suggested to be an ideal set of genes for data normalization across all life stages, while GAPDH was proven to be a poor reference gene [ 35 ].…”

Section: Discussionmentioning

confidence: 99%

Reference gene stability of a synanthropic fly, Chrysomya megacephala

et al. 2015

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BackgroundStable reference genes are essential for accurate normalization in gene expression studies with reverse transcription quantitative polymerase chain reaction (qPCR). A synanthropic fly, Chrysomya megacephala, is a well known medical vector and forensic indicator. Unfortunately, previous studies did not look at the stability of reference genes used in C. megacephala.ResultsIn this study, the expression level of Actin, ribosomal protein L8 (Rpl8), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1), α-tubulin (α-TUB), β-tubulin (β-TUB), TATA binding box (TBP), 18S rRNA (18S) and ribosomal protein S7 (Rps7) were evaluated for their stability using online software RefFinder, which combines the normal software of the ΔCt method, BestKeeper, Normfinder, and geNorm. Moreover the number of suitable reference gene pairs was also suggested by Excel-based geNorm. The expression levels of these reference genes were evaluated under different experimental conditions with special perspectives of forensic applications: developmental stages (eggs, first, second and third instar larvae, pupae and adults); food sources of larvae (pork, fish and chicken); feeding larvae with drugs (untreated control, Estazolam and Marvelon); feeding larvae with heavy metals (untreated control, cadmium and zinc); tissues of adults (head, thorax, abdomen, legs and wings). According to RefFinder, EF1 was the most suitable reference gene of developmental stages, food and tissues; 18S and GAPDH were the most suitable reference genes for drugs and heavy metals, respectively, which could be widely used for quantification of target gene expression with qPCR in C. megacephala. Suitable reference gene pairs were also suggested by geNorm.ConclusionThis fundamental but vital work should facilitate the gene studies of related biological processes and deepen the understanding in physiology, toxicology, and especially medical and forensic entomology of C. megacephala.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1175-9) contains supplementary material, which is available to authorized users.

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“…MNE is the ratio of the mean of the reference and target genes cycle thresholds ( Ct s), including efficiency values: MNE = ( E reference ) Ctreference ∕( E target ) Cttarget . All mRNA levels were normalized using the mean of the reference genes Gapdh and Rp49 , both validated in a previous study (Cardoso et al, 2014). …”

Section: Methodsmentioning

confidence: 99%

Evolution of genes involved in feeding preference and metabolic processes in Calliphoridae (Diptera: Calyptratae)

Cardoso

Marinho

Monfardini

et al. 2016

PeerJ

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BackgroundThe genotype-phenotype interactions among traits governing feeding preference are of fundamental importance to behavioral genetics and evolutionary biology. The genetic basis of behavioral traits has been explored in different taxa using different approaches. However, the complex nature of the genetic mechanisms undergirding behavior is poorly understood. Here, we present an evolutionary study of candidate genes related to parasitism in Calliphoridae (Diptera: Calyptratae). Closely related species in this family exhibit distinct larval feeding habits, most notably necro-saprophagy and obligate parasitism.MethodsTo understand the genetic and molecular bases underlying these habits, expression levels of eight candidate genes for feeding behavior—Cyp6g2, foraging, glutamate dehydrogenase, Jonah65aiv, Malvolio, PGRP-SC2, RPS6-p70-protein kinase, and smooth—were measured in four species using qPCR. Moreover we used expression values and sequence information to reconstruct the relationship among species and the dN/dS rate to infer possible sites under selection.ResultsFor most candidate genes, no statistically significant differences were observed, indicating a high degree of conservation in expression. However, Malvolio was differentially expressed between habits. Evolutionary analyses based on transcript levels and nucleotide sequences of Malvolio coding region suggest that transcript levels were correlated to feeding habit preferences among species, although deviations under a strictly neutral model were also observed in statistical tests.DiscussionMalvolio was the only gene demonstrating a possible connection to feeding habit. Differences in gene expression may be involved in (or be a result of) the genetic regulation of Calliphoridae feeding habit. Our results are the first steps towards understanding the genetic basis and evolution of feeding behavior in Calliphoridae using a functional approach.

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Selection and validation of reference genes for functional studies in the Calliphoridae family

Cited by 15 publications

References 40 publications

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

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

Reference gene stability of a synanthropic fly, Chrysomya megacephala

Evolution of genes involved in feeding preference and metabolic processes in Calliphoridae (Diptera: Calyptratae)

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