Selection of Stable Reference Genes for Real-Time Quantitative PCR Analysis in Edwardsiella tarda

Sun, Zhongyang; Deng, Jia; Wu, Haizhen; Wang, Qiyao; Zhang, Yuanxing

doi:10.4014/jmb.1605.05023

Cited by 12 publications

(4 citation statements)

References 21 publications

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“…Thus, in this study, rrs would be used for normalization to validate the selection result in 12% ethanol. Furthermore, in order to neutralize the impact of the high abundance of rrs , the templates for rrs should be diluted more times than the samples for the other genes in RT-qPCR analysis, however the different dilution ratios can lead to more human errors (Sun et al, 2016 ). Thus, in each experiment, three independent biological replicates and three technical replicates were required at least.…”

Section: Discussionmentioning

confidence: 99%

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization Under Ethanol Stress Conditions in Oenococcus oeni SD-2a

et al. 2018

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The powerful Quantitative real-time PCR (RT-qPCR) was widely used to assess gene expression levels, which requires the optimal reference genes used for normalization. Oenococcus oeni (O. oeni), as the one of most important microorganisms in wine industry and the most resistant lactic acid bacteria (LAB) species to ethanol, has not been investigated regarding the selection of stable reference genes for RT-qPCR normalization under ethanol stress conditions. In this study, nine candidate reference genes (proC, dnaG, rpoA, ldhD, ddlA, rrs, gyrA, gyrB, and dpoIII) were analyzed to determine the most stable reference genes for RT-qPCR in O. oeni SD-2a under different ethanol stress conditions (8, 12, and 16% (v/v) ethanol). The transcript stabilities of these genes were evaluated using the algorithms geNorm, NormFinder, and BestKeeper. The results showed that dnaG and dpoIII were selected as the best reference genes across all experimental ethanol conditions. Considering single stress experimental modes, dpoIII and dnaG would be suitable to normalize expression level for 8% ethanol shock treatment, while the combination of gyrA, gyrB, and rrs would be suitable for 12% ethanol shock treatment. proC and gyrB revealed the most stable expression in 16% ethanol shock treatment. This study selected and validated for the first time the reference genes for RT-qPCR normalization in O. oeni SD-2a under ethanol stress conditions.

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Section: Discussionmentioning

confidence: 99%

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization Under Ethanol Stress Conditions in Oenococcus oeni SD-2a

et al. 2018

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“…GyrA is responsible for DNA cleavage and ligation, and GyrB contains ATP-binding sites [46]. DNA gyrase subunits have been recommended as reference genes in several bacterium species, including Oenococcus oeni [47], Shewanella psychrophila [36], Dwardsiella tarda [48], Xanthomonas fragariae [42], Bacillus velezensis [49], Corynebacterium pseudotuberculosis [19] and Herbaspirillum rubrisubalbicans [50].…”

Section: Discussionmentioning

confidence: 99%

Reference Genes for Expression Analyses by qRT-PCR in Enterobacter cancerogenus

Pan,

Zhao,

Zeng

et al. 2024

Microorganisms

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The Enterobacter cancerogenus strain EcHa1 was isolated from the dead larvae of Helicoverpa armigera, and has the potential for biocontrol of some Lepidoptera insects. In order to screen insecticidal-related genes by qRT-PCR, stable endogenous reference genes used for normalizing qRT-PCR data were selected and evaluated from 13 housekeeping genes (HKGs). The expression levels of the HKGs were determined using qRT-PCR under different experimental conditions, including two culture temperatures and three bacterial OD values. Five stability analysis methods (Ct, BestKeeper, NormFinder, geNorm, and RefFinder) were used to comprehensively rank the candidate genes. The results showed that the optimal reference genes varied under different experimental conditions. The combination of gyrA and gyrB was recommended as the best reference gene combination at 28 °C, while gyrA and rpoB was the best combination at 37 °C. When the OD values were 0.5, 1.0 and 2.0, the recommended reference gene combinations were ftsZ and gyrA, rpoB and gyrB, and gyrA and pyk, respectively. The most suitable reference genes were gyrA and gyrB under all experimental conditions. Using gyrA and gyrB as the reference genes for qRT-PCR, EcHa1 was found to invade all tissues of the H. armigera larvae, and expressed a candidate pathogenic factor Hcp at high levels in gut, Malpighian tubules, and epidermis tissues. This study not only establishes an accurate and reliable normalization for qRT-PCR in entomopathogenic bacteria but also lays a solid foundation for further study of functional genes in E. cancerogenus.

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“…A number of housekeeping genes such as 16S ribosomal RNA (16S rRNA), ribosome protein L13 (rbL13), RNA polymerase factor, DNA polymerase factor and elongation factor have been used as reference genes in different expression profiling studies in bacteria [11][12][13][14]. However, it is reported that the expression of housekeeping genes may vary among various species and different experimental conditions.…”

Section: Introductionmentioning

confidence: 99%

rpoB and efp are stable candidate reference genes for quantitative real-time PCR analysis in Saccharopolyspora spinosa

Liu¹,

Zhang²,

Huang³

et al. 2021

Biotechnology & Biotechnological Equipment

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Spinosad (spinosyn A and spinosyn D), the secondary metabolite produced by Saccharopolyspora spinosa, is a potent insecticide with low effects on the environment and mammals. Strategies such as metabolic engineering, mutagenesis and fermentation process optimization have been employed for its production enhancement. Quantitative real-time polymerase chain reaction(qRT-PCR) is one of the preferred methods for the evaluation of gene transcript levels, whose accuracy and sensitivity depend on the normalization using optimal reference genes. However, no single reference gene is universally appropriate for all strains under various conditions. In this study, the transcriptional stability of 35 candidate reference genes, including 23 traditionally used reference genes and 12 novel reference genes identified in S. spinosa homologous species, was analysed in S. spinosa ATCC 49460 and spinosad highyield strain S. spinosa S3-3 under three fermentation phases. The transcriptional stability of these genes was assessed by three statistical algorithms, geNorm, NormFinder and BestKeeper. The overall rankings suggested that rpoB and efp were the most stable candidate reference genes, and they may be the most promising reference genes for further study on the measurement of expression levels of target genes involved in the biosynthetic process of spinosad.

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Selection of Stable Reference Genes for Real-Time Quantitative PCR Analysis in Edwardsiella tarda

Cited by 12 publications

References 21 publications

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization Under Ethanol Stress Conditions in Oenococcus oeni SD-2a

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Normalization Under Ethanol Stress Conditions in Oenococcus oeni SD-2a

Reference Genes for Expression Analyses by qRT-PCR in Enterobacter cancerogenus

rpoB and efp are stable candidate reference genes for quantitative real-time PCR analysis in Saccharopolyspora spinosa

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