Identification and Validation of Reference Genes for Quantitative Real-Time PCR in Drosophila suzukii (Diptera: Drosophilidae)

Zhai, Yifan; Lin, Qingsong; Zhou, Xianhong; Zhang, Xiaoyan; Liu, Tingli; Yu, Yong

doi:10.1371/journal.pone.0106800

Cited by 65 publications

(70 citation statements)

References 31 publications

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“…In Drosophila , it has been shown that a larger panel of reference genes is required as the sample size and inherent complexity grows616263. Selection of gene types is also crucial.…”

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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“…In Drosophila , it has been shown that a larger panel of reference genes is required as the sample size and inherent complexity grows616263. Selection of gene types is also crucial.…”

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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“…), and Drosophila suzukii (Zhai et al . ). In particular, in Drosophila melanogaster , the expression stability of several housekeeping genes has been evaluated in various sample conditions, such as injury, heat stress, different diets, different ages, and neurodegeneration (Ling & Salvaterra ; Ponton et al .…”

Section: Introductionmentioning

confidence: 97%

“…However, considering the high sensitivity of PCR, appreciable errors in data interpretation can occur when using qRT‐PCR to determine the transcript levels of target genes among various sample conditions, such as different tissues, treatments, amount of extracted RNA, and PCR amplification efficiencies (Ling & Salvaterra ; Zhai et al . ; Moon et al . , ).…”

Section: Introductionmentioning

confidence: 99%

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Validation of reference genes for quantitative real‐time polymerase chain reaction in Drosophila melanogaster exposed to two chemicals

Kim

et al. 2019

Entomological Research

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Drosophila melanogaster is attracted to chemicals produced by fermentation and it is abundantly found in rotten fruits. Considering its habitat, the fruit fly is reported to be tolerant to environmental chemicals. Quantitative real‐time polymerase chain reaction was employed to investigate the expression pattern and physiological function of genes putatively involved in chemical detoxification. In quantitative real‐time polymerase chain reaction assays, normalization of target gene expression with internal reference genes is required. These reference genes should be stably expressed during chemical exposure and in chemical‐free conditions. In this study, therefore, we used two programs (geNorm and BestKeeper) to evaluate the expression stability of five reference genes (nd, rpL18, ef1β, hsp22 and tbp) in female adult flies exposed to various concentrations of methanol and ethyl acetate. Four genes (nd, rpL18, ef1β and tbp) were found to be suitable for use as reference genes in methanol‐treated flies and three genes (ef1β, nd, tbp) were found to be suitable for use as reference genes in ethyl acetate‐treated flies. These results suggested that a combination of two genes among these stably expressed genes can be used for accurate normalization of target gene expression in quantitative real‐time polymerase chain reaction‐based determination of gene expression profiles in D. melanogaster treated with both chemicals.

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“…In the Tephritidae family there are two studies on the oriental fruit fly, Bactrocera dorsalis 1718 and one in the West Indian fruit fly Anastrepha obliqua 19. Among other dipteran species, there are three studies on Drosophila melanogaster 152021 and a single one on each of D. suzukii 22, Musca domestica 23, Lucilia cuprina 24 and the Calliphoridae family25. Interestingly, there are no studies published on any mosquito species.…”

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confidence: 99%

Housekeeping in Tephritid insects: the best gene choice for expression analyses in the medfly and the olive fly

Sagri

Koskinioti

Gregoriou

et al. 2017

Sci Rep

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Real-time quantitative-PCR has been a priceless tool for gene expression analyses. The reaction, however, needs proper normalization with the use of housekeeping genes (HKGs), whose expression remains stable throughout the experimental conditions. Often, the combination of several genes is required for accurate normalization. Most importantly, there are no universal HKGs which can be used since their expression varies among different organisms, tissues or experimental conditions. In the present study, nine common HKGs (RPL19, tbp, ubx, GAPDH, α-TUB, β-TUB, 14-3-3zeta, RPE and actin3) are evaluated in thirteen different body parts, developmental stages and reproductive and olfactory tissues of two insects of agricultural importance, the medfly and the olive fly. Three software programs based on different algorithms were used (geNorm, NormFinder and BestKeeper) and gave different ranking of HKG stabilities. This confirms once again that the stability of common HKGs should not be taken for granted and demonstrates the caution that is needed in the choice of the appropriate HKGs. Finally, by estimating the average of a standard score of the stability values resulted by the three programs we were able to provide a useful consensus key for the choice of the best HKG combination in various tissues of the two insects.

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Identification and Validation of Reference Genes for Quantitative Real-Time PCR in Drosophila suzukii (Diptera: Drosophilidae)

Cited by 65 publications

References 31 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

Validation of reference genes for quantitative real‐time polymerase chain reaction in Drosophila melanogaster exposed to two chemicals

Housekeeping in Tephritid insects: the best gene choice for expression analyses in the medfly and the olive fly

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