Suitable reference genes for RT-qPCR analysis in Dichelops melacanthus (Hemiptera: Pentatomidae)

Pinheiro, Daniele H.; Moreira, Raquel Oliveira; Leite, Natália Alves; Redoan, A. C.; Xavier, André da Silva; Barros, B. de A.; Carneiro, N. P.

doi:10.1007/s11033-020-05550-z

Cited by 14 publications

(9 citation statements)

References 61 publications

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“…Thus, RNA quality and amplification were of sufficient quality to be used in RT-qPCR. Our RNA quality and amplification results agree with other reference gene validation studies conducted on the other insects (Shakeel et al, 2015;Pinheiro et al, 2020).…”

Section: Discussionsupporting

confidence: 89%

“…However, extreme care must be taken to avoid erroneous results ( Liang et al, 2014 ). One of the most common strategies for correcting experimental errors introduced during the steps of RT-qPCR analysis is the normalization of RT-qPCR data with reference genes ( Pinheiro et al, 2020 ). Inappropriate reference gene selection can obscure or magnify real biological changes caused by changes in reference gene expression ( Zhu et al, 2014 ).…”

Section: Discussionmentioning

confidence: 99%

“…Consideration of the significance and diverse specificity of reference genes, many reference gene sets have been validated in various insect species, such as Spodoptera frugiperda ( Zhou et al, 2021 ), Rhopalosiphum padi ( Li M. et al, 2021 ), Aquatica leii ( Fu and Meyer-Rochow, 2021 ), Tuta absoluta ( Yan et al, 2021 ), Dichelops melacanthus ( Pinheiro et al, 2020 ), Thermobia domestica ( Bai et al, 2020 ), Apolygus lucorum ( Luo et al, 2020 ), Lymantria dispar ( Yin et al, 2020 ), Drosophila melanogaster ( Kim et al, 2020 ), Phenacoccus solenopsis ( Zheng et al, 2019 ), Chilo partellus ( Adeyinka et al, 2019 ), Harmonia axyridis ( Yang et al, 2018 ), Liriomyza trifolii ( Chang et al, 2017 ), Myzus persicae ( Kang et al, 2017 ), and B. odoriphaga ( Shi et al, 2016 ) under various experimental conditions. However, a universal reference gene has not yet been identified.…”

Section: Introductionmentioning

confidence: 99%

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Selection and Validation of Reference Genes for RT-qPCR Normalization in Bradysia odoriphaga (Diptera: Sciaridae) Under Insecticides Stress

Huang

Chen

et al. 2022

Front. Physiol.

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Bradysia odoriphaga (Diptera: Sciaridae) is the most serious root maggot pest which causes substantial damage to the Chinese chive. Organophosphate (OP) and neonicotinoid insecticides are widely used chemical pesticides and play important roles in controlling B. odoriphaga. However, a strong selection pressure following repeated pesticide applications has led to the development of resistant populations of this insect. To understand the insecticide resistance mechanism in B. odoriphaga, gene expression analysis might be required. Appropriate reference gene selection is a critical prerequisite for gene expression studies, as the expression stability of reference genes can be affected by experimental conditions, resulting in biased or erroneous results. The present study shows the expression profile of nine commonly used reference genes [elongation factor 1α (EF-1α), actin2 (ACT), elongation factor 2α (EF-2α), glucose-6-phosphate dehydrogenase (G6PDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), ubiquitin-conjugating enzyme (UBC), and α-tubulin (TUB)] was systematically analyzed under insecticide stress. Moreover, we also evaluated their expression stability in other experimental conditions, including developmental stages, sexes, and tissues. Five programs (NormFinder, geNorm, BestKeeper, RefFinder, and ΔCt) were used to validate the suitability of candidate reference genes. The results revealed that the most appropriate sets of reference genes were RPL10 and ACT across phoxim; ACT and TUB across chlorpyrifos and chlorfluazuron; EF1α and TUB across imidacloprid; EF1α and EF2α across developmental stages; RPL10 and TUB across larvae; EF1α and ACT across tissues, and ACT and G6PDH across sex. These results will facilitate the standardization of RT-qPCR and contribute to further research on B. odoriphaga gene function under insecticides stress.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Selection and Validation of Reference Genes for RT-qPCR Normalization in Bradysia odoriphaga (Diptera: Sciaridae) Under Insecticides Stress

Huang

Chen

et al. 2022

Front. Physiol.

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“…qPCR has become the most commonly used tool in molecular biology for the quantitative analysis of the expression and transcription of insect target genes. Despite that the basis of a successful qPCR experiment is the adoption of appropriate reference genes (Hunter & Garrels 1977;Tao et al 2019), there are virtually no publicly available reference genes with a stable expression level under all experimental conditions for insects (Li et al 2013;Ladror et al 2014;Yang et al 2014;Singh et al 2019;Pinheiro et al 2020;Wang et al 2020b). Therefore, it is necessary to screen for reference genes that can be used for quantitative expression studies under different experimental conditions.…”

Section: Discussionmentioning

confidence: 99%

“…ΔCt method First, the ΔCt method was used to analyze the Ct values of all candidate reference genes, and the standard deviations of those values were compared (Silver et al 2006;Pinheiro et al 2020;Wang et al 2020a). The ΔCt method ranked 28S rRNA as the most stably expressed reference gene and RPL10 as the least stable among the three developmental stages (Fig.…”

Section: Expression Stability Of the Candidate Reference Genesmentioning

confidence: 99%

Selection and evaluation of reference genes for quantitative real‐time PCR analysis in lac insect (Kerria lacca)

Wang

Ling

Bashir

et al. 2022

Entomological Research

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The screening and identification of suitable reference genes are key steps in experiments involving the quantitative real-time polymerase chain reaction. However, the reference genes of Kerria lacca, a lac insect with considerable economic significance, have not been investigated. In this study, we used five statistical algorithms (ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder) to evaluate the stability of eight candidate reference genes (18S rRNA, 28S rRNA, β-ACT, EF1-α, RPL10, RPL13, RPS11, and RPS15) in different developmental stages and tissues of K. lacca and under dsRNA treatment. Our results showed that the most stable reference genes were 28S rRNA and RPS11 for the developmental stages, RPL13 and RPS11 for different tissues, and β-ACT and EF1-α for dsRNA treatments. Although the target genes FAS and 17β-HSD showed consistent expression profiles (alone or in combination) when normalized to the expression levels of the stable genes, those changed when the least stable reference gene (18S rRNA) was used. These results highlight that suitable reference genes should be chosen according to the specific experimental conditions used. The reliability and stability of different reference genes can provide a firm foundation for gene expression analysis in lac insects.

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Role of EPG in Developing and Assessing Control Methods for Heteropteran Crop Pests

Panizzi

Lucini

Mitchell

2021

Entomology in Focus

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Suitable reference genes for RT-qPCR analysis in Dichelops melacanthus (Hemiptera: Pentatomidae)

Cited by 14 publications

References 61 publications

Selection and Validation of Reference Genes for RT-qPCR Normalization in Bradysia odoriphaga (Diptera: Sciaridae) Under Insecticides Stress

Selection and Validation of Reference Genes for RT-qPCR Normalization in Bradysia odoriphaga (Diptera: Sciaridae) Under Insecticides Stress

Selection and evaluation of reference genes for quantitative real‐time PCR analysis in lac insect (Kerria lacca)

Role of EPG in Developing and Assessing Control Methods for Heteropteran Crop Pests

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