Validation of differential gene expression in muscle engineered from rat groin adipose tissue by quantitative real-time PCR

An, Yang; Reimers, Kerstin; Allmeling, Christina; Liu, Jieli; Lazaridis, Andrea; Vogt, Peter M.

doi:10.1016/j.bbrc.2012.04.073

Cited by 12 publications

(12 citation statements)

References 21 publications

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“…Currently, however, many common reference genes are used as internal controls without any evaluation of their variance and instability, and most publications only use a single internal control for normalization. These un-validated single reference genes, however, were proved to be not always reliable under various experimental conditions [12]–[16]. Therefore, more and more biologists pay their attention to the selection and validation of reliable reference gene(s) expressed stably regardless of different experimental conditions from the species they are interested in, in order to avoid unnecessary errors in qRT-PCR analysis.…”

Section: Discussionmentioning

confidence: 99%

“…The most common normalization method is to compare the mRNA level of the target gene with that of a reference gene whose expression level is considered stable regardless of different experimental conditions [7]–[9]. However, none of the reference genes discovered thus far is consistently expressed in a universal and invariant way under various experimental conditions [10], [11], and recent reference gene selection studies indicate that a single reference gene is generally insufficient to normalize the expression data of all target genes [12]–[16].…”

Section: Introductionmentioning

confidence: 99%

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Expression Profiling in Bemisia tabaci under Insecticide Treatment: Indicating the Necessity for Custom Reference Gene Selection

et al. 2014

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Finding a suitable reference gene is the key for qRT-PCR analysis. However, none of the reference gene discovered thus far can be utilized universally under various biotic and abiotic experimental conditions. In this study, we further examine the stability of candidate reference genes under a single abiotic factor, insecticide treatment. After being exposed to eight commercially available insecticides, which belong to five different classes, the expression profiles of eight housekeeping genes in the sweetpotato whitefly, Bemisia tabaci, one of the most invasive and destructive pests in the world, were investigated using qRT-PCR analysis. In summary, elongation factor 1α (EF1α), α-tubulin (TUB1α) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified as the most stable reference genes under the insecticide treatment. The initial assessment of candidate reference genes was further validated with the expression of two target genes, a P450 (Cyp6cm1) and a glutathione S-transferase (GST). However, ranking of reference genes varied substantially among intra- and inter-classes of insecticides. These combined data strongly suggested the necessity of conducting custom reference gene selection designed for each and every experimental condition, even when examining the same abiotic or biotic factor.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Expression Profiling in Bemisia tabaci under Insecticide Treatment: Indicating the Necessity for Custom Reference Gene Selection

et al. 2014

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“…In other study, also did not find stable results for Actb gene from hypothalamus of an obesity rat model [45]. Actb and Gapdh genes were rejected in muscle tissue by qBase, software that uses M-Value to analyze reference genes [26]. In our study, Actb and Gapdh genes were shown to be stable in all tissues analyzed by three different software; the only exception was for mesenteric fat analysis through geNorm, which rejected both genes, along with Hprt1.…”

Section: Genorm Analysismentioning

confidence: 84%

“…In hypothalamus the most stable gene was Actb when comparing two are different, a third reference gene must be used, and, if they are similar, it is not necessary to evaluate a third gene [26]. Dheda et al [27] demonstrated after three experiments with different reference genes that the results can be significantly different from those obtained when an invalidated reference gene is used.…”

Section: Genorm Analysismentioning

confidence: 97%

Validation of Reference Genes for qPCR Analysis of Resistance Training and Androgenic Anabolic Steroids on Hypothalamus, Adrenal Gland and Fat Tissue

Pozzi¹,

Le²,

Fern³

et al. 2016

J Steroids Horm Sci

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Background: Real-time quantitative Polymerase Chain Reaction (qPCR) is a technique used for quantification of gene expression and the use of reference genes is very important to normalize the quantification results. Aim:To validate the most suitable reference genes for resistance exercise training (REx) and use of nandrolone decanoate (DECA) in three different rat tissues. Methods:A total of 40 adult male Wistar rats were distributed into four groups: exposed to vehicle three times per week (wk) (CT); eight wk of REx exposed to vehicle three times per wk (T); exposed to DECA three times per wk (D); eight wk of REx exposed to DECA three times per wk (TD). Stability of the following genes was evaluated: beta actin (Actb), alpha Tubulin (Tubulin), Glyceraldehyde-3-phosphate dehydrogenase (Gapdh), Hypoxanthine phosphoribosyltransferase-1 (Hprt1) and 18s Ribossomal RNA (18s) in hypothalamus, adrenal gland and mesenteric fat tissue using GeNorm, NormFinder and BestKeeper software. Results:In hypothalamus and adrenal, all genes were suitable and none was rejected by statistical analysis; however, in fat tissue, Actb, Gapdh and Hprt1 genes were rejected by geNorm but not the others two software. Conclusion:In hypothalamus and adrenal all selected genes analized were stable and can be used for qPCR gene expression analysis. However, in fat tissue we suggest the Tubulin gene as most stable gene.

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“…Use of a single housekeeping gene without validation of its stability can lead to a greater than 20-fold error in calculations of gene expression 22. During recent years, a growing number of studies of reference gene suitability have been conducted in humans 29-31, other mammals 32, 33, plants 34-38 as well as arthropods (Table S4). With more and more genomes and transcriptomes available for arthropods, there are at least 45 arthropod species that have been investigated for reference gene identification and selection under diverse experimental conditions.…”

Section: Discussionmentioning

confidence: 99%

Selection of Reference Genes for Expression Studies of Xenobiotic Adaptation in Tetranychus urticae

et al. 2016

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Quantitative real-time PCR (qRT-PCR) is an extensively used, high-throughput method to analyze transcriptional expression of genes of interest. An appropriate normalization strategy with reliable reference genes is required for calculating gene expression across diverse experimental conditions. In this study, we aim to identify the most stable reference genes for expression studies of xenobiotic adaptation in Tetranychus urticae, an extremely polyphagous herbivore causing significant yield reduction of agriculture. We chose eight commonly used housekeeping genes as candidates. The qRT-PCR expression data for these genes were evaluated from seven populations: a susceptible and three acaricide resistant populations feeding on lima beans, and three other susceptible populations which had been shifted host from lima beans to three other plant species. The stability of the candidate reference genes was then assessed using four different algorithms (comparative ΔCt method, geNorm, NormFinder, and BestKeeper). Additionally, we used an online web-based tool (RefFinder) to assign an overall final rank for each candidate gene. Our study found that CycA and Rp49 are best for investigating gene expression in acaricide susceptible and resistant populations. GAPDH, Rp49, and Rpl18 are best for host plant shift studies. And GAPDH and Rp49 were the most stable reference genes when investigating gene expression under changes in both experimental conditions. These results will facilitate research in revealing molecular mechanisms underlying the xenobiotic adaptation of this notorious agricultural pest.

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Validation of differential gene expression in muscle engineered from rat groin adipose tissue by quantitative real-time PCR

Cited by 12 publications

References 21 publications

Expression Profiling in Bemisia tabaci under Insecticide Treatment: Indicating the Necessity for Custom Reference Gene Selection

Expression Profiling in Bemisia tabaci under Insecticide Treatment: Indicating the Necessity for Custom Reference Gene Selection

Validation of Reference Genes for qPCR Analysis of Resistance Training and Androgenic Anabolic Steroids on Hypothalamus, Adrenal Gland and Fat Tissue

Selection of Reference Genes for Expression Studies of Xenobiotic Adaptation in Tetranychus urticae

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