Identification of suitable qPCR reference genes in leaves of Brassica oleracea under abiotic stresses

Brulle, Franck; Bernard, Fabien; Vandenbulcke, Franck; Cuny, Damien; Dumez, Sylvain

doi:10.1007/s10646-014-1209-7

Cited by 27 publications

(18 citation statements)

References 64 publications

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“…As noted previously, CYP was the worst reference gene in a diverse pool of poplar [32], however the optimal stability of CYP expression identified in our study was in agreement with the result of Petunia hybrid transcriptomic analysis [33]. Additionally, the observations on the reference genes ACT , UBQ and RPL2 with stable expression in peach (Rosaceae) [34], Brassica oleracea [35] and tomato [28], respectively, had been completely verified in our studies. It has been reported that ELFA was the most stable in potato during biotic and abiotic stress [36], but poorly ranked during light stress in tomato [28].…”

Section: Discussionsupporting

confidence: 92%

Selection of Reference Genes for Gene Expression Studies in Siberian Apricot (Prunus sibirica L.) Germplasm Using Quantitative Real-Time PCR

et al. 2014

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Quantitative real time reverse transcription polymerase chain reaction has been applied in a vast range of studies of gene expression analysis. However, real-time PCR data must be normalized with one or more reference genes. In this study, eleven putative consistently expressed genes (ACT, TUA, TUB, CYP, DNAj, ELFA, F-box27, RPL12, GAPDH, UBC and UBQ) in nine Siberian Apricot Germplasms (including much variability) were evaluated for their potential as references for the normalization of gene expression by NormFinder and geNorm programs. From our studies, ACT, UBC, CYP, UBQ and RPL12 as suitable for normalization were identified by geNorm, while UBC and CYP as the best pair by NormFinder. Moreover, UBC was selected as the most stably expressed gene by both algorithms in different Siberian Apricot seed samples. We also detected that a set of three genes (ACT, CYP and UBC) by geNorm as control for normalization could lead to accurate results. Furthermore, the expression levels of oleosin gene were analyzed to validate the suitability of the selected reference genes. These obtained experimental results could make an important contribution to normalize real-time PCR data for gene expression analysis in Siberian Apricot Germplasm.

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Section: Discussionsupporting

confidence: 92%

Selection of Reference Genes for Gene Expression Studies in Siberian Apricot (Prunus sibirica L.) Germplasm Using Quantitative Real-Time PCR

et al. 2014

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“…To estimate the expression stability of reference genes, we measured the relative quantities of crossing point values of genes obtained using the formula of Hellemans et al [30]. The geometric mean of the 3 most stable reference genes in cupric condition was used to calculate expression of target gene levels according to Brulle et al [35]. The procedures of the analyses strictly followed the manuals of BestKeeper, version 1 [32], NormFinder, version 5 [33], and geNorm, version 3.5 [34] algorithms.…”

Section: Rna Isolation Cdna Synthesis Housekeeping Genes Validationmentioning

confidence: 99%

“…The average of cycle threshold (Ct) values and expression levels of each candidate reference gene in 10 different date palm hypocotyl stressed samples were transferred to software, as fully described by Brulle et al [35]. The average of cycle threshold (Ct) values and expression levels of each candidate reference gene in 10 different date palm hypocotyl stressed samples were transferred to software, as fully described by Brulle et al [35].…”

Section: Internal Reference Gene Validation For Qpcr Analysismentioning

confidence: 99%

“…The procedures of the analyses strictly followed the manuals of BestKeeper, version 1 [32], NormFinder, version 5 [33], and geNorm, version 3.5 [34] algorithms. The geometric mean of the 3 most stable reference genes in cupric condition was used to calculate expression of target gene levels according to Brulle et al [35]. Absolute quantification of gene expression levels are in log2.…”

Section: Rna Isolation Cdna Synthesis Housekeeping Genes Validationmentioning

confidence: 99%

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Copper toxicity and date palm (Phoenix dactylifera) seedling tolerance: Monitoring of related biomarkers

Chaâbene

Hakim

Rorat

et al. 2017

Enviro Toxic and Chemistry

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Date palm (Phoenix dactylifera) seeds were exposed to different copper (Cu) solutions to examine plant stress responses. Low Cu concentrations (0.02 and 0.2 mM) caused an increase of seed germination, whereas higher Cu amounts (2 mM) significantly inhibited seed germination, delayed hypocotyl elongation, increased seedling mortality, and reduced the germination index by more than 90%. Metal-related toxicity symptoms appeared after 15 d of 2 mM of Cu exposure. Biochemical activities such as amylase activity and redox balance elements were examined to study the relationship between external Cu amount and internal plant response. The present study showed that amylolytic activity was dose- and time-dependent. Likewise, H O production increased after exposure to Cu, which was correlated with thiobarbituric acid reactive substance (TBARS) accumulation. Furthermore at low Cu concentrations, superoxide dismutase (SOD) and catalase (CAT) activities increased, suggesting that date palm seed stimulated its metal homeostasis networks. However, the highest cupric ion amounts increased cell oxidant accumulation and reduced enzyme production. Gene expression level measures of P. dactylifera phytochelatin synthase (Pdpcs) and P. dactylifera metallothionein (Pdmt) encoding genes have been carried out to investigate the implication of PdPCS and PdMT proteins in Cu homeostasis and/or its sequestration. Phoenix dactylifera metallothionein induction reached a peak after 30 d of exposure to 0.2 mM of Cu. However, it was down-regulated in plants exposed to higher Cu concentrations. In the same conditions, Pdpcs was overexpressed during 1 mo of exposure before it decreased thereafter. These observations provide a new insight into date palm cell response to Cu, a metal that can be toxic but that is also an essential element. Environ Toxicol Chem 2018;37:797-806. © 2017 SETAC.

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“…In addition, HKGs, such as actin (ACT), b-tubulin (TUB), GAPDH, polyubiquitin, and elongation factor 1-a (EF1-a) (Bustin 2002;Radonic et al 2004;Czechowski et al 2005;Gutierrez et al 2008b), whose protein products are supposed to have stable and uniform expression across different tissues and developmental stages, are commonly used as internal reference genes (Paolacci et al 2009). However, recent studies have suggested that the expression of these genes can vary significantly under different environmental conditions (Thellin et al 1999;Schmittgen and Zakrajsek 2000;Suzuki et al 2000;Brulle et al 2014). Therefore, it is necessary to identify the internal reference gene(s) for the efficient quantification of a target mRNA in a given set of biological samples by qRT-PCR.…”

Section: Introductionmentioning

confidence: 99%

Evaluation and selection of reference genes for ecotoxicogenomic study of the green alga Closterium ehrenbergii using quantitative real-time PCR

et al. 2015

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The green alga Closterium ehrenbergii occurs in fresh water environments and has been suggested as a model for ecotoxicological assessment. Quantitative real-time PCR (qRT-PCR), with its high sensitivity and specificity, is a preferred method for reliable quantification of gene expression levels. qRT-PCR requires reference genes to normalize the transcription level of the target gene, and selection of appropriate references is crucial. Here, we evaluated nine housekeeping genes, that is, 18S rRNA, ACT, TUA, TUB, eIF, H4, UBQ, rps4, and GAPDH, using 34 RNA samples of C. ehrenbergii cultured in various environments (e.g. exposure to heat shock, UV, metals, and non-metallic chemicals). Each housekeeping gene tested displayed different ranges of C T values for each experimental condition. The gene stability was determined using the descriptive statistic software geNorm, which showed that ACT, H4, and TUA were the most suitable reference genes for all the conditions tested. In addition, at least three genes were required for proper normalization. With these references, we assessed the expression level of the heat shock protein 70 (HSP70) gene in C. ehrenbergii cells exposed to thermal and toxic contaminant stress and found that it was significantly up-regulated by these stressors. This study provides potential reference genes for gene expression studies on C. ehrenbergii with qRT-PCR.

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Identification of suitable qPCR reference genes in leaves of Brassica oleracea under abiotic stresses

Cited by 27 publications

References 64 publications

Selection of Reference Genes for Gene Expression Studies in Siberian Apricot (Prunus sibirica L.) Germplasm Using Quantitative Real-Time PCR

Selection of Reference Genes for Gene Expression Studies in Siberian Apricot (Prunus sibirica L.) Germplasm Using Quantitative Real-Time PCR

Copper toxicity and date palm (Phoenix dactylifera) seedling tolerance: Monitoring of related biomarkers

Evaluation and selection of reference genes for ecotoxicogenomic study of the green alga Closterium ehrenbergii using quantitative real-time PCR

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