Selection and Validation of Potential Reference Genes for Quantitative Real-Time PCR Analysis in Blaptica Dubia (Serville, 1838) (Blattidae, Blaberidae) (original) (raw)
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Bilge International Journal of Science and Technology Research, 2022
Quantitative RT-PCR (q-RT-PCR) is a powerful tool that allows large-scale analysis of very small changes in gene expression. For the calculation of gene expression, such as the delta-delta Ct method, different PCR primer efficiencies (E) may affect the result, as PCR primer yields are assumed to be comparable for the gene of interest and housekeeping gene. Therefore, identification of a suitable reference gene for data normalization is an important step in the development of qPCR assays. Furthermore, accurate and reliable results depend on the use of stable reference genes for normalization. The aim of the current study is the identification and validation of a set of six housekeeping genes (GADPH, RPS18, α-TUB, EF1α, ArgK, and ACTB) in cockroach species Nauphoeta cinerea adults using five different algorithms (ΔCt method, Bestkeeper, geNorm, Normfinder and RefFinder) to evaluate the stability of selected reference genes expression. Our results show that α-Tub use provides accurate normalization of gene expression levels in N. cinerea adults. In addition, since the GADPH is selected as the second most stable reference gene, GADPH can be also used for transcript analysis N. cinerea adults. Our study also showed that ACTB (β-actin) should not be used for normalizing transcript levels when examining N. cinerea adults. Additionally, validation studies for reference genes in cockroaches are very few (only one) in the literature. Therefore, the results highlight the need for validation of reference genes under biotic and abiotic conditions in q-RT-PCR studies in cockroaches.
BMC research notes, 2013
Quantitative RT-PCR (q-RT-PCR) is a powerful tool that allows for the large scale analysis of small changes in gene expression. Accurate and reliable results depend on the use of stable reference genes for normalization. However, the expression of some widely used housekeeping genes can vary under different experimental setups. To our knowledge, no validation studies have been reported for reference genes in cockroaches. The aim of the current study is the identification and validation of a set of eight housekeeping genes during the first gonadotrophic cycle of the cockroach, Diploptera punctata. This study made use of two different algorithms (geNorm and Normfinder) to evaluate the stability of gene expression. Candidate housekeeping genes were sequenced: β-actin (Actin), elongation factor 1 alpha (EF1a), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), armadillo (Arm), ribosomal protein L32 (RpL32), succinate dehydrogenase (SDHa), annexin IX (AnnIX) and α-tubulin (Tub). The expre...
Frontiers in Physiology
Cockroaches are important global urban pests from aesthetic and health perspectives. Insecticides represent the most cost-effective way to control cockroaches and limit their impacts on human health. However, cockroaches readily develop insecticide resistance, which can quickly limit efficacy of even the newest and most effective insecticide products. The goal of this research was to understand whole-body physiological responses in German cockroaches, at the metatranscriptome level, to defined insecticide selection pressures. We used the insecticide indoxacarb as the selecting insecticide, which is an important bait active ingredient for cockroach control. Six generations of selection with indoxacarb bait produced a strain with substantial (>20×) resistance relative to inbred control lines originating from the same parental stock. Metatranscriptome sequencing revealed 1,123 significantly differentially expressed (DE) genes in ≥two of three statistical models (81 upregulated and 1...
Scientific reports, 2017
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...
Journal of experimental zoology. Part B, Molecular and developmental evolution, 2018
The German cockroach, Blattella germanica, is a worldwide pest that infests buildings, including homes, restaurants, and hospitals, often living in unsanitary conditions. As a disease vector and producer of allergens, this species has major health and economic impacts on humans. Factors contributing to the success of the German cockroach include its resistance to a broad range of insecticides, immunity to many pathogens, and its ability, as an extreme generalist omnivore, to survive on most food sources. The recently published genome shows that B. germanica has an exceptionally high number of protein coding genes. In this study, we investigate the functions of the 93 significantly expanded gene families with the aim to better understand the success of B. germanica as a major pest despite such inhospitable conditions. We find major expansions in gene families with functions related to the detoxification of insecticides and allelochemicals, defense against pathogens, digestion, sensor...
Journal of Virological Methods, 2010
Quantitative real-time reverse transcription-PCR (qRT-PCR) has been used widely to measure gene transcription regulation in cells. qRT-PCR must include one or more internal housekeeping genes to normalize data collection. A strategy to use the host cell 28S rRNA as a housekeeping gene in qRT-PCR analysis of gene transcription of insect cells infected by baculovirus and ascovirus was developed. It has been found that the 28S rRNA reverse primer can be incorporated in the oligo-dT-primed cDNA synthesis reaction. In such a way, amplification of 28S cDNA showed lower and less variable cycle thresholds in cells infected by viruses than by using only oligo-dT and other published housekeeping genes such as the TATA box binding protein (TBP) gene, the peptidyl prolyl isomerase A (PPI) gene and the ribosomal protein 13 (L13) gene. Incorporation of the 28S reverse primer in oligo-dT-primed cDNA synthesis also does not interfere with the detection of other polymerase II transcribed genes.
BMC Molecular Biology
Genomic studies measuring transcriptional responses to changing environments and stress currently make their way into the field of evolutionary ecology and ecotoxicology. To investigate a small to medium number of genes or to confirm large scale microarray studies, Quantitative Reverse Transcriptase PCR (QRT-PCR) can achieve high accuracy of quantification when key standards, such as normalization, are carefully set. In this study, we validated potential reference genes for their use as endogenous controls under different chemical and physical stresses in two species of soil-living Collembola, Folsomia candida and Orchesella cincta. Treatments for F. candida were cadmium exposure, phenanthrene exposure, desiccation, heat shock and pH stress, and for O. cincta cadmium, desiccation, heat shock and starvation.
Journal of Insect Science, 2008
In this study an important and often neglected aspect of gene expression studies in insects, the validation of appropriate reference genes with stable expression levels between sample groups, is addressed. Although in this paper the reference gene selection for the honeybee, Apis mellifera L. (Hymenoptera: Apidae) head was tested in the context of bacterial challenge with Escherichia coli, this work can serve as a resource to help select and screen insect reference genes for gene expression studies in any tissue and under any experimental manipulation. Since it is recommended to use multiple reference genes for accurate normalization, we analyzed the expression of eleven candidate reference genes in the honeybee head, for their potential use in the analysis of differential gene expression following bacterial challenge. Three software programs, BestKeeper, Normfinder and geNorm, were used to assess candidate reference genes. GeNorm recommended the use of four reference genes. Both geNorm and Normfinder identified the genes GAPDH, RPS18, actin and RPL13a as the most stable ones, only differing in their ranking order. BestKeeper identified RPS18 as being the reference gene with the least overall variation, but also actin and GAPDH were found to be the second and third most stable expressed gene. By a combination of three software programs the genes actin, RPS18 and GAPDH were found suitable reference genes in the honeybee head in the context of bacterial infection.
Evaluation of reference genes for insect olfaction studies
Parasites & vectors, 2015
Quantitative reverse transcription PCR (qRT-PCR) is a robust and accessible method to assay gene expression and to infer gene regulation. Being a chain of procedures, this technique is subject to systematic error due to biological and technical limitations mainly set by the starting material and downstream procedures. Thus, rigorous data normalization is critical to grant reliability and repeatability of gene expression quantification by qRT-PCR. A number of 'housekeeping genes', involved in basic cellular functions, have been commonly used as internal controls for this normalization process. However, these genes could themselves be regulated and must therefore be tested a priori. We evaluated eight reference genes for their stability as internal controls of olfactory gene expression in the antennae of Rhodnius prolixus, a Chagas disease vector. Five experimental conditions, including changes in age, developmental stage and feeding status were tested in both sexes. We show t...
Journal of medical entomology, 2004
Exposure and sensitization to cockroach allergens is an important risk factor for allergic disease in humans. Despite a recent burgeoning of clinical and socioeconomic studies regarding environmental pervasiveness and human exposure to cockroach allergens, little is known about the basic biology of these proteins. The purpose of this study was to ascertain gene expression patterns and the tissue distribution of Blattella germanica allergen 1 (Bla g 1), a perennial indoor environmental allergen, thought to be involved in digestion in cockroaches. We also investigated the relative potential contribution of different life stages of the German cockroach to environmental Bla g 1. Enzyme-linked immunosorbent assay was used to quantify the Bla g 1 contents of feces and various anatomical tissues, and Northern blot analysis was used to elucidate tissue-speciÞc expression of Bla g 1. Results showed that the Bla g 1 protein is most prevalent in the midgut, and the Bla g 1 gene is exclusively expressed by midgut cells. Although Bla g 1 is produced by both sexes and all life stages of the German cockroach, adult females produce and excrete signiÞcantly more Bla g 1 in their feces than males or nymphs, even when corrected for body mass or mass of voided feces. Our results show that the concentration of Bla g 1 in feces of adult females is 6-to 7-and 30-fold higher than in adult males and nymphs, respectively, probably because females process more food than other life stages of the German cockroach.