Evaluation of Reference Genes for Quantitative Polymerase Chain Reaction across Life Cycle Stages and Tissue Types of <i>Tribolium castaneum</i>

Toutges, Michelle; Hartzer, Kris; Lord, Jeffrey C.; Oppert, Brenda

doi:10.1021/jf101603j

Cited by 55 publications

(37 citation statements)

References 8 publications

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“…For each cDNA sample the PCR reactions were conducted in triplicate and relative target gene expression was normalized to that of YQE_05788, which encodes ribosomal protein S3P. Ribosomal protein S3 is a relatively more stable normalizing gene for qRT-PCR in another beetle, Tribolium castaneum , compared to the more routinely used actin or tubulin genes [29]. Fold change was calculated for each normalized gene in relation to the expression of the unfed male treatment using the 2 -ΔΔCT method [30].…”

Section: Methodsmentioning

confidence: 99%

Comparative transcriptomics of mountain pine beetle pheromone-biosynthetic tissues and functional analysis of CYP6DE3

et al. 2017

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BackgroundThe mountain pine beetle (MPB, Dendroctonus ponderosae Hopkins) is a highly destructive pest of pine forests in western North America. During flight to a new host tree and initiation of feeding, mountain pine beetles release aggregation pheromones. The biosynthetic pathways of these pheromones are sex-specific and localized in the midgut and fat body, but the enzymes involved have not all been identified or characterized.ResultsWe used a comparative RNA-Seq analysis between fed and unfed male and female MPB midguts and fat bodies to identify candidate genes involved in pheromone biosynthesis. The 13,407 potentially unique transcripts showed clear separation based on feeding state and gender. Gene co-expression network construction and examination using petal identified gene groups that were tightly connected. This, as well as other co-expression and gene ontology analyses, identified all four known pheromone biosynthetic genes, confirmed the tentative identification of four others from a previous study, and suggested nine novel candidates. One cytochrome P450 monooxygenase, CYP6DE3, identified as a possible exo-brevicomin-biosynthetic enzyme in this study, was functionally characterized and likely is involved in resin detoxification rather than pheromone biosynthesis.ConclusionsOur analysis supported previously characterized pheromone-biosynthetic genes involved in exo-brevicomin and frontalin biosynthesis and identified a number of candidate cytochrome P450 monooxygenases and a putative cyclase for further studies. Functional analyses of CYP6DE3 suggest its role in resin detoxification and underscore the limitation of using high-throughput data to tentatively identify candidate genes. Further functional analyses of candidate genes found in this study should lead to the full characterization of MPB pheromone biosynthetic pathways and the identification of molecular targets for possible pest management strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3696-4) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Comparative transcriptomics of mountain pine beetle pheromone-biosynthetic tissues and functional analysis of CYP6DE3

et al. 2017

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“…HSP90 expression profiles were determined using forward primer 5′-CCT CAA GTC CAC GCA TCC AG-3′ and reverse primer 5′-TCG CCT CCT TGT GCA TCT TC-3′, which were designed using the Primer3 software (Rozen and Skaletsky 2000; http://frodo.wi.mit.edu) according to the HSP90 sequence (NP_001094067) and purchased from Sigma-Aldrich Chemie (Taufkirchen, Germany). The T. castaneum housekeeping gene encoding ribosomal protein RPS3 was used as an internal control (Toutges et al 2005). Quantitative real-time RT-PCR was carried out on a CFX 96 Real-Time PCR Detection System (Bio-Rad) using the SsoFast™ EvaGreen® Supermix (Bio-Rad).…”

Section: Phylogenetic Analysismentioning

confidence: 99%

Post-embryonic functions of HSP90 in Tribolium castaneum include the regulation of compound eye development

Knorr

Vilcinskas

2011

Dev Genes Evol

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Heat shock protein 90 (HSP90) belongs to a family of conserved chaperons with multiple roles in stress adaptation and development, including spermatogenesis, oogenesis and embryogenesis in insects. In the red flour beetle, Tribolium castaneum, we found that HSP90 is transiently upregulated during larval development, in prepupae, in female pupae and in adults, suggesting multiple post-embryonic roles. We found that silencing HSP90 expression by RNA interference was lethal within 10 days at all developmental stages. Titration experiments revealed that larvae were more susceptible than pupae or beetles. Interestingly, HSP90 silencing in final instar larvae resulted in abnormal pupal phenotypes lacking compound eyes and exhibiting prepupal features, suggesting developmental arrest at the prepupal stage. Our results suggest that HSP90 functions can be expanded beyond the known ones in insect embryogenesis to include roles in post-embryonic development such as the regulation of compound eye development.

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“…Reverse transcription quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive, accurate and widely used method for gene expression analysis in order to understand biological processes and physiological functions, as well as for validation of the results of microarray analysis and other techniques [1], [2]. One of the critical challenges of qRT-PCR analysis for reliable mRNA quantification in any biological system is the availability of appropriate normalization genes, the expression level of which is considered stable, regardless of cell type and across various experimental conditions [3].…”

Section: Introductionmentioning

confidence: 99%

Identification and Validation of Reference Genes for Gene Expression Analysis Using Quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae)

et al. 2013

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Reverse transcription quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantification of gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes need to show stable expression under the given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in Spodoptera litura. In this study, eight candidate reference genes, elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), beta actin (ACTB), beta FTZ-F1 (FTZF1), ubiquinol-cytochrome c reductase (UCCR), and arginine kinase (AK), were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs, BestKeeper, geNorm and Normfinder, and the comparative ΔCt method. We determined the expression levels of the candidate reference genes for three biotic factors (developmental stage, tissue and population), and four abiotic treatments (temperature, insecticide, food and starvation). The results indicated that the best sets of candidates as reference genes were as follows: GAPDH and UCCR for developmental stages; RPL10, AK and EF1 for different tissues; RPL10 and EF1 for different populations in China; GAPDH and EF1 for temperature-stressed larvae; AK and ACTB for larvae treated with different insecticides; RPL10, GAPDH and UCCR for larvae fed different diets; RPS3 and ACTB for starved larvae. We believe that these results make an important contribution to gene analysis studies in S. litura and form the basis of further research on stable reference genes in S. litura and other organisms.

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Evaluation of Reference Genes for Quantitative Polymerase Chain Reaction across Life Cycle Stages and Tissue Types of Tribolium castaneum

Cited by 55 publications

References 8 publications

Comparative transcriptomics of mountain pine beetle pheromone-biosynthetic tissues and functional analysis of CYP6DE3

Comparative transcriptomics of mountain pine beetle pheromone-biosynthetic tissues and functional analysis of CYP6DE3

Post-embryonic functions of HSP90 in Tribolium castaneum include the regulation of compound eye development

Identification and Validation of Reference Genes for Gene Expression Analysis Using Quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae)

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