Validation of reference genes for gene expression studies in non viruliferous and viruliferous Frankliniella occidentalis (Thysanoptera: Thripidae)

Quantitative real-time PCR (qRT-PCR) is a powerful technique for measuring and evaluating gene expressions during different biological processes. To facilitate gene expression studies, normalization with respect to stable housekeeping genes (HKGs) is mandatory. The western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae), the main vector of Tomato spotted wilt virus (TSWV), is a very destructive invasive species. In this study, expression profiles of 11 candidate HKGs, including β-actin (Actin), α-tubulin (Tubulin), elongation factor 1 α (EF1A), vacuolar-typeH+-ATPase (ATPase), NADH-ubiquinone oxidoreductase (NADH), heat shock protein 60 (HSP60), heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), ribosomal protein l32 (RPL32), 28S ribosomal RNA (28S), and 18S ribosomal RNA (18S), from no nviruliferous and viruliferous F. occidentalis were investigated. Four distinct algorithms, geNorm, Normfinder, BestKeeper, and the ΔCt method, were employed to determine the performance of these genes as endogenous controls under the virus condition. Based on RefFinder, which integrates all four analytical algorithms to compare and rank the candidates, HSP70 , HSP60, EF1A, and RPL32 were the most stable housekeeping genes. This work is the initial first step to establish a standardized qRT-PCR analysis in F. occidentalis. Additionally, this study lays a foundation for the research in the interactions between TSWV and F. occidentalis.

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Gene expression studies in different genotypes of an ectomycorrhizal fungus require a high number of reliable reference genes

10.7287/peerj.preprints.2125v1 ◽

2016 ◽

Author(s):

Joske Ruytinx ◽

Tony Remans ◽

Jan V Colpaert

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Intraspecific Variability ◽

Standard Technique ◽

Housekeeping Genes ◽

Ectomycorrhizal Fungus ◽

Suillus Luteus ◽

Qrt Pcr ◽

Expression Studies ◽

Gene Expression Studies

Quantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the expression analysis of a set of chosen genes of interest. The accuracy and reliability of qRT-PCR measurements strongly depends on the normalization with appropriate endogenous reference genes. In this study a set of candidate reference genes for the use in gene expression studies of a basidiomycete fungus, Suillus luteus, exposed to toxic concentrations of zinc or cadmium was identified, evaluated and validated. Seven candidate genes were selected from cDNA-AFLP as stably expressed and the algorithms geNorm and Normfinder were used to evaluate these genes alongside the traditionally used housekeeping genes (actin, tubulin) in different S. luteus isolates. The use of several S. luteus isolates revealed that each isolate has its own most stably expressed set of reference genes, regardless of the metal treatments, in casu metal exposures. Metal treatments had only a minor impact on the expression of the candidate reference genes. The validated reference genes outperform the in fungal research commonly used single, arbitrary chosen (“housekeeping”) genes in terms of reliability, and have the potential to be suitable reference genes when studying the effect of other environmental factors. A relatively high number of reference genes is required to correct for intraspecific variability when studying natural populations.

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Gene expression studies in different genotypes of an ectomycorrhizal fungus require a high number of reliable reference genes

10.7287/peerj.preprints.2125 ◽

2016 ◽

Author(s):

Joske Ruytinx ◽

Tony Remans ◽

Jan V Colpaert

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Intraspecific Variability ◽

Standard Technique ◽

Housekeeping Genes ◽

Ectomycorrhizal Fungus ◽

Suillus Luteus ◽

Qrt Pcr ◽

Expression Studies ◽

Gene Expression Studies

Quantitative reverse transcription PCR (qRT-PCR) has become the standard technique for the expression analysis of a set of chosen genes of interest. The accuracy and reliability of qRT-PCR measurements strongly depends on the normalization with appropriate endogenous reference genes. In this study a set of candidate reference genes for the use in gene expression studies of a basidiomycete fungus, Suillus luteus, exposed to toxic concentrations of zinc or cadmium was identified, evaluated and validated. Seven candidate genes were selected from cDNA-AFLP as stably expressed and the algorithms geNorm and Normfinder were used to evaluate these genes alongside the traditionally used housekeeping genes (actin, tubulin) in different S. luteus isolates. The use of several S. luteus isolates revealed that each isolate has its own most stably expressed set of reference genes, regardless of the metal treatments, in casu metal exposures. Metal treatments had only a minor impact on the expression of the candidate reference genes. The validated reference genes outperform the in fungal research commonly used single, arbitrary chosen (“housekeeping”) genes in terms of reliability, and have the potential to be suitable reference genes when studying the effect of other environmental factors. A relatively high number of reference genes is required to correct for intraspecific variability when studying natural populations.

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Evaluation of qPCR reference genes for taimen (Hucho taimen) under heat stress

Scientific Reports ◽

10.1038/s41598-021-03872-x ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Xiaoxing Yang ◽

Guangxiang Tong ◽

Le Dong ◽

Ting Yan ◽

Huan Xu ◽

...

Keyword(s):

Gene Expression ◽

Heat Stress ◽

Heat Shock ◽

Heat Shock Protein ◽

Ribosomal Protein ◽

Ribosomal Rna ◽

Reference Genes ◽

28S Rrna ◽

Qrt Pcr ◽

Hucho Taimen

AbstractAs a powerful and attractive method for detecting gene expression, qRT-PCR has been broadly used in aquaculture research. Understanding the biology of taimen (Hucho taimen) has drawn increasing interest because of its ecological and economic value. Stable reference genes are required for the reliable quantification of gene expression, but such genes have not yet been optimized for taimen. In this study, the stability levels of 10 commonly used candidate reference genes were evaluated using geNorm, NormFinder, BestKeeper, and RefFinder. The expression levels of the 10 genes were detected using 240 samples from 48 experimental groups consisting of 40 individuals treated under four heat-stress conditions (18, 20, 22, and 24 °C) for 24 h and 26 °C for 4, 24, 48, and 72 h. Six tissues (blood, heart, brain, gill, skin, and liver) were collected from each individual. Ribosomal protein S29 (RPS29) and ribosomal protein L19 (RPL19) were the most stable genes among all of the samples, whereas 28S ribosomal RNA (28S rRNA), attachment region binding protein (ARBP), and 18S ribosomal RNA (18S rRNA) were the least stable. These results were verified by an expression analysis of taimen heat-stress genes (heat shock protein 60, hsp60, and heat shock protein 70, hsp70). In conclusion, RPS29 and RPL19 are the optimal reference genes for qRT-PCR analyses of taimen, irrespective of the tissue and experimental conditions. These results allow the reliable study of gene expression in taimen.

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Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

BioMed Research International ◽

10.1155/2015/363575 ◽

2015 ◽

Vol 2015 ◽

pp. 1-20 ◽

Cited By ~ 6

Author(s):

Cora S. Thiel ◽

Swantje Hauschild ◽

Svantje Tauber ◽

Katrin Paulsen ◽

Christiane Raig ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Molecular Mechanisms ◽

Expression Profiles ◽

Housekeeping Genes ◽

Altered Gravity ◽

Experimental Conditions ◽

Expression Levels ◽

Expression Studies ◽

Gene Expression Studies

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes”) are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

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Assessment of common housekeeping genes as reference for gene expression studies using RT-qPCR in mouse choroid plexus

Scientific Reports ◽

10.1038/s41598-021-82800-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kim Hoa Ho ◽

Annarita Patrizi

Keyword(s):

Gene Expression ◽

Choroid Plexus ◽

Sensory Deprivation ◽

Housekeeping Genes ◽

Housekeeping Gene ◽

Experimental Conditions ◽

Brain Repair ◽

Expression Studies ◽

Testing Algorithms ◽

Gene Expression Studies

AbstractChoroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.

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