Selection and validation of reference genes for qRT-PCR analysis during biological invasions: The thermal adaptability of Bemisia tabaci MED

Dai, Tian-Mei; Lu, Zengkui; Liu, Wan‐Xue; Wan, Fanghao

doi:10.1371/journal.pone.0173821

Cited by 32 publications

(29 citation statements)

References 77 publications

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“…Although β-ACT and ArgK are traditional reference genes and have been used in many gene expression studies, they are not always stably expressed. Consistent with our results, many studies have shown unstable expression of β-ACT under variable conditions in a variety of species, including Bemisia tabaci 40 , M. separata 35 , F. occidentalis 39 , Henosepilachna vigintioctopunctata 41 , P. japonica 38 , Hippodamia convergens 42 and C. maculata 30 , as well as for ArgK in Spodoptera litura 43 , P. japonica 38 , C. maculata 30 and E. onukii 8 .…”

Section: Experimental Conditionsupporting

confidence: 92%

Selection of reference genes for normalization of RT-qPCR data in gene expression studies in Anthonomus eugenii Cano (Coleoptera: Curculionidae)

Pinheiro

Siegfried

2020

Sci Rep

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The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is the main insect pest of peppers (Capsicum spp.) throughout the southern U.S. and a potential target for novel control methods that may require gene expression analyses. careful selection of adequate reference genes to normalize Rt-qpcR data is an important prerequisite for gene expression studies since the expression stability of reference genes can be affected by the experimental conditions leading to biased or erroneous results. the lack of studies on validation of reference genes for Rt-qpcR analysis in A. eugenii limits the investigation of gene expression, therefore it is needed a systematic selection of suitable reference genes for data normalization. In the present study, three programs (BestKeeper, geNorm and normfinder) were used to analyze the expression stability of candidate reference genes (β-ACT, ArgK, EF1-α, GAPDH, RPL12, RPS23, α-TUB, 18S and 28S) in A. eugenii under different experimental conditions. our results revealed that the most stably expressed reference genes in A. eugenii varied according to the experimental condition evaluated: developmental stages (EF1-α, 18S and RPL12), sex (RPS23 and RPL12), low temperature (GAPDH and α-TUB), high temperature (α-TUB and RPS23), all temperatures (α-TUB and GAPDH), starvation (RPL12 and α-TUB), and dsRNA exposure (α-TUB and RPL12). Our study provides for the first time valuable information on appropriate reference genes that can be used in the analysis of gene expression by Rt-qpcR in biological experiments involving A. eugenii.Reverse-transcription quantitative PCR (RT-qPCR) is widely used in gene expression studies due to its simplicity, reproducibility, high sensitivity, accuracy and cost-effectiveness 1,2 . Although RT-qPCR is considered a highly accurate technique, several experimental factors can lead to results that are not reliable measurements of gene expression. These factors include purity and integrity of RNA, quantity of starting RNA and cDNA, reverse transcription and PCR efficiency, and pipetting errors 3 . Thus, in RT-qPCR analysis is necessary to use reference genes to normalize the data in order to eliminate or at least reduce the technical variation among the tested samples and precisely estimate the expression of the target genes 4 .Usually, housekeeping genes related to basic cellular functions are used as reference genes in the normalization strategy because these genes are supposed to have constitutive and stable expression under a variety of physiological conditions and experimental treatments. However, several studies have demonstrated that the expression of housekeeping genes is not always stable and can be influenced by developmental stage, tissue, sex, and biotic or abiotic stresses that the organism is subjected 5-9 . Therefore, the selection of suitable reference genes according to the specific experimental conditions is essential to ensure accurate results.The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is the most economica...

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Section: Experimental Conditionsupporting

confidence: 92%

Selection of reference genes for normalization of RT-qPCR data in gene expression studies in Anthonomus eugenii Cano (Coleoptera: Curculionidae)

Pinheiro

Siegfried

2020

Sci Rep

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“…Production of dsRNA transcription templates and dsRNA synthesis dsRNA targeting MED Dnmt3 and eGFP was synthesised via PCR and in vitro transcription as described by Dai et al (2017). PCR using specific primers with a T7 RNA polymerase promoter generated a product that was subsequently employed as a template for in vitro transcription using a MEGAscriptT7 High Yield Transcription Kit (Ambion, Austin, TX, USA).…”

Section: Bioinformatics Characterization Of the Btdnmt3 Genementioning

confidence: 99%

“…All amplifications were confirmed via sequencing, and the specificity of qRT-PCR was estimated through melting curve analysis. Beta-1-tubulin (b-tub) and elongation factor 1 alpha (EF1-a) were used as internal controls to normalize the relative expression of Dnmt mRNA (Dai et al, 2017). A reaction volume of 20 ll was employed, containing 1.0 ml cDNA template, 10.0 ml of 23 TransStart TM Green qPCR SuperMix (Transgen), 0.3 ml of each primer (10 lM), 0.4 ml passive reference dye and 8.0 ll doubledistilled H 2 O.…”

Section: Qrt-pcrmentioning

confidence: 99%

Molecular characterizations of DNA methyltransferase 3 and its roles in temperature tolerance in the whitefly, Bemisia tabaci Mediterranean

Dai

Wang

et al. 2017

Insect Molecular Biology

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The Bemisia tabaci Mediterranean (MED) cryptic species is an invasive pest, distributed worldwide, with high ecological adaptability and thermotolerance. DNA methylation (a reversible chromatin modification) is one possible change that may occur within an organism subjected to environmental stress. To assess the effects of temperature stress on DNA methyltransferase 3 (Dnmt3) in MED, we cloned and sequenced BtDnmt3 and identified its functions in response to high and low temperatures. The fulllength cDNA of BtDnmt3 was 3913 bp, with an open reading frame of 1962 bp, encoding a 73.89 kDa protein. In situ hybridization showed that BtDnmt3 was expressed mainly in the posterior region. BtDnmt3 messenger RNA expression levels were significantly down-regulated after exposure to heat shock and significantly up-regulated after exposure to cold shock. Furthermore, after feeding on double-stranded RNA specific for BtDnmt3, both heat resistance and cold resistance were significantly decreased, suggesting that BtDnmt3 is associated with thermal stress response and indicating a differential response to high-and low-temperature stress in MED. Together, these results highlight a potential role for DNA methylation in thermal resistance, which is a process important to successful invasion and colonization of an alien species in various environments.

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“…; Dai et al . ). Therefore, it is necessary to evaluate the stability of reference genes under specific experimental conditions.…”

Section: Introductionmentioning

confidence: 97%

“…Housekeeping genes have been conventionally used for reference genes in RT-qPCR analysis, including 18S rRNA, Actin, Tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor-1 (EF-1), ribosomal protein (RP) and TATA binding protein (TBP) (Ponton et al 2011;Zhong et al 2013;Avicor et al 2014;Cardoso et al 2014;Zhai et al 2014). Nevertheless, some studies have reported that the expression of universal reference genes can show great variation across different tissues and developmental stages and under different experimental treatments Collins et al 2014;Dai et al 2017). Therefore, it is necessary to evaluate the stability of reference genes under specific experimental conditions.…”

Section: Introductionmentioning

confidence: 99%

Selection of reference genes for quantitative real‐time polymerase chain reaction normalization inBradysia odoriphaga(Diptera: Sciaridae)

Tang

Dai

Zhang

2019

Entomological Science

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Selecting stable reference genes for reverse transcription quantitative real‐time polymerase chain reaction analysis is critically important for gene expression. Bradysia odoriphaga is the most serious pest of Chinese chives, but our knowledge of its genetics is limited. In the present study, five housekeeping genes (β‐Actin, α‐Tubulin, RPS7, GAPDH and 18S rRNA) were cloned from B. odoriphaga using the combined techniques of reverse transcription polymerase chain reaction with rapid amplification of cDNA ends. The five genes, together with RPS15, were quantified for transcription stability in B. odoriphaga under various experimental conditions. Their expression stability was evaluated using four different algorithms (a comparative ΔCt method, GeNorm, NormFinder and BestKeeper). Additionally, we used an online Web‐based tool (RefFinder) to assign an overall final rank to each candidate gene. These analyses identified 18S, RPS15 and RPS7 as the most stable reference genes across developmental stages. 18S, RPS7 and Tubulin were the most stable reference genes for monitoring gene expression across different tissues of adults. RPS7, GAPDH and Tubulin were selected as the best reference genes across different larval tissues. GAPDH and RPS15 were selected to normalize gene expression for experiments of insecticide treatments. Actin and GAPDH are considered suitable reference genes in experiments of temperature treatments. Actin and Tubulin are considered suitable reference genes for starvation experiments. This study provides an important supplement of suitable reference genes for B. odoriphaga and these results provide clues toward the understanding of the genes’ biological functions in B. odoriphaga.

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Selection and validation of reference genes for qRT-PCR analysis during biological invasions: The thermal adaptability of Bemisia tabaci MED

Cited by 32 publications

References 77 publications

Selection of reference genes for normalization of RT-qPCR data in gene expression studies in Anthonomus eugenii Cano (Coleoptera: Curculionidae)

Selection of reference genes for normalization of RT-qPCR data in gene expression studies in Anthonomus eugenii Cano (Coleoptera: Curculionidae)

Molecular characterizations of DNA methyltransferase 3 and its roles in temperature tolerance in the whitefly, Bemisia tabaci Mediterranean

Selection of reference genes for quantitative real‐time polymerase chain reaction normalization inBradysia odoriphaga(Diptera: Sciaridae)

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