Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis

Chen, Chun; Xie, Tingna; Ye, Sudan; Jensen, Annette Bruun; Eilenberg, Jørgen

doi:10.1016/j.bjm.2015.11.031

Cited by 19 publications

(17 citation statements)

References 42 publications

(51 reference statements)

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“…Nonetheless, research has shown that the expression levels of such stably expressed genes may not be stable in various cell types, at different developmental stages or under different experimental conditions [ 14 – 17 ]. Therefore, the evaluation and selection of reference genes under different experimental conditions are of great significance for obtaining reliable and precise qRT-PCR results [ 18 , 19 ].…”

Section: Introductionmentioning

confidence: 99%

Reference Gene Selection for Quantitative Real-Time PCR of Mycelia fromLentinula edodesunder High-Temperature Stress

Zhao

Yang

Chen

et al. 2018

BioMed Research International

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Housekeeping genes are important for measuring the transcription expression of functional genes; 10 traditional reference genes, TUB, TUA, GADPH, EF1, 18S, GTP, ACT, UBI, UBC, and H2A, were tested for their adequacy in Lentinula edodes (L. edodes). Using specific primers, mRNA levels of these candidate housekeeping genes were evaluated in mycelia of L. edodes, which were treated with high-temperature stress at 37°C for 0, 4, 8, 12, 18, and 24 hours. After treatment, expression stability of candidate genes was evaluated using three statistical software programs: geNorm, NormFinder, and BestKeeper. According to geNorm, TUB had the lowest M values in L. edodes strains 18 and 18N44. Using NormFinder, the best candidate reference gene in strain 18 was TUB (0.030), and the best candidate reference gene in strain 18N44 was UBI (0.047). In BestKeeper analysis, the standard deviation (SD) values of UBC, TUA, H2A, EF1, ACT, 18S, and GTP in strain 18 and those of GADPH and GTP in strain 18N44 were greater than 1; thus, these genes were disqualified as reference genes. Taken together, only UBI and TUB were found to be desirable reference genes by BestKeeper software. Based on the results of three software analyses, TUB was the most stable gene under all conditions and was verified as an appropriate reference gene for quantitative real-time polymerase chain reaction in L. edodes mycelia under high-temperature stress.

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Section: Introductionmentioning

confidence: 99%

Reference Gene Selection for Quantitative Real-Time PCR of Mycelia fromLentinula edodesunder High-Temperature Stress

Zhao

Yang

Chen

et al. 2018

BioMed Research International

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“…Furthermore, expression analysis of pathogen genes during infection and colonization in the host plant has been the main step in elucidating the biological function of pathogen genes. To reduce errors in expression quantification of pathogen genes caused by the adaptability of the pathogen, as well as nutrition and stress in host plants [ 11 , 12 ], pathogen PCR reference genes have been included in investigations. However, there is growing evidence showing there is no single, universal gene that could be utilized in various experimental conditions [ 13 ], and the stability of reference genes should be validated before these are used for normalization of gene expression [ 14 , 15 ].…”

Section: Introductionmentioning

confidence: 99%

Identification and evaluation of PCR reference genes for host and pathogen in sugarcane-Sporisorium scitamineum interaction system

et al. 2018

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BackgroundSugarcane (Saccharum L. plant) is an important crop for sugar and bio-energy production around the world. Among sugarcane diseases, smut caused by Sporisorium scitamineum is one of the major fungal diseases causing severe losses to the sugarcane industry. The use of PCR reference genes is essential to the normalization of data on gene expression involving the sugarcane-S. scitamineum interaction system; however, no report that addresses criteria in selecting these reference genes has been published to date.ResultsIn this study, 10 sugarcane genes and eight S. scitamineum genes were selected as candidate PCR reference genes in the sugarcane-S. scitamineum interaction system. The stability and reliability of these 18 candidate genes were analyzed in smut-resistant (NCo376) and -susceptible (YC71–374) genotypes using the statistical algorithms geNorm, NormFinder, BestKeeper, and deltaCt method. Subsequently, the relative expression levels of the sugarcane chitinase I-3 gene and S. scitamineum chorismate mutase gene were determined to validate the applicability of these sugarcane and S. scitamineum PCR reference genes, respectively. We finally found that the acyl-CoA dehydrogenase gene (ACAD), serine/arginine repetitive matrix protein 1 gene (SARMp1), or their combination (ACAD + SARMp1) could be utilized as the most suitable reference genes for normalization of sugarcane gene expression in sugarcane bud tissues after S. scitamineum infection. Similarly, the inosine 5′-monophosphate dehydrogenase gene (S10), the SEC65-signal recognition particle subunit gene (S11), or their combination (S10 + S11) were suitable for normalization of S. scitamineum gene expression in sugarcane bud tissues.ConclusionsThe PCR reference genes ACAD, SARMp1, S10, and S11 may be employed in gene transcriptional studies involving the sugarcane-S. scitamineum interaction system.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4854-z) contains supplementary material, which is available to authorized users.

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“…Furthermore, GAPDH variable expression gave false negative results re-IL4 expression levels in tuberculosis patients (Dheda et al, 2004). 18S is another internal control which was chosen to do the same relative assay instead of GAPDH, since its RNA subunits are the most stable housekeeping gene during different types of nutritional stresses in plants (Chen et al, 2016) or during long-term exposure to salts (Wang et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

GAPDH spike RNA as an alternative for housekeeping genes in relative gene expression assay using real-time PCR

Atwan

2020

Bull Natl Res Cent

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Background and aim of study: qPCR is a robust technique which quantifies the expressions of target genes in relation to reference genes. Stresses such as virus infection or heat shock change expressions of many cellular genes including the reference genes, so the aim was to introduce a constant calibrator to normalize the data to. Methodology: Constructed glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plasmid was transcribed to GAPDH RNA and used as spike RNA. Spiked RNA samples were subjected to qPCR at different conditions such as virus infection, IFN treatment, or mild heat shock.The results: Adenovirus hexon in interferon-deficient cells showed different expression levels when data were normalized to GAPDH or 18S. Consistently, hexon expression levels were different in untreated cells under the control or heat-shocked conditions when data were normalized to GAPDH or 18S. Promyelocytic leukemia protein II (PML-II) expression level was lower in HeLa-PML-II-deficient cells (PML-II-Kd) compared to the control when the data were normalized to GAPDH as a reference gene and also in GAPDH RNA spiked, which showed reasonable consistency. More consistent data were obtained when the GAPDH normalizer was added before the step of treating the extracted RNA with DNase compared to add it after the treatment or directly to the qPCR reaction. Conclusion:The internal controls that were chosen for this study completely changed the experimental results since they were affected with the experimental conditions. However, GAPDH spike RNA level was stable in its amplification at different kinds of stresses. So it can be an alternative for housekeeping gene due to its stability at these different conditions.

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Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis

Cited by 19 publications

References 42 publications

Reference Gene Selection for Quantitative Real-Time PCR of Mycelia fromLentinula edodesunder High-Temperature Stress

Reference Gene Selection for Quantitative Real-Time PCR of Mycelia fromLentinula edodesunder High-Temperature Stress

Identification and evaluation of PCR reference genes for host and pathogen in sugarcane-Sporisorium scitamineum interaction system

GAPDH spike RNA as an alternative for housekeeping genes in relative gene expression assay using real-time PCR

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