Reference gene selection for quantitative real-time reverse-transcriptase PCR in orchardgrass subjected to various abiotic stresses

Huang, Linkai; Yan, Haidong; Jiang, Xueyu; Zhang, Yu; Zhang, Xinquan; Yang, Jicheng; Zeng, Bing; Xu, Bin; Yin, Guohua; Lee, Samantha; Yan, Yanhong; Ma, Xiao; Yan, Peng

doi:10.1016/j.gene.2014.10.017

Cited by 41 publications

(32 citation statements)

References 49 publications

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“…Therefore it is important to use at least three different softwares in order to achieve best results as possible in particular to avoid selection of co-regulated genes (Jacob et al, 2013). GeNorm, based on the pairwise variations with no consideration of a possible co-regulation, is particularly sensitive concerning this aspect (Huang et al, 2014). The 12 reference genes were ranked according to the results from the three algorithms.…”

Section: Discussionmentioning

confidence: 99%

“…They are crucial for data normalization and will determine the reliability of the data and of their interpretation (Bustin et al, 2009). Good reference genes are stably expressed under chosen experimental conditions and should have a level of expression comparable to those of the target genes (Gimeno et al, 2014; Huang et al, 2014). The key difference between a reference gene and a real housekeeping gene is that a housekeeping gene should be stably expressed whatever the conditions, at every stage of the plant life, in every tissues.…”

Section: Introductionmentioning

confidence: 99%

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Selection and validation of reference genes for quantitative real-time PCR analysis of gene expression in Cichorium intybus

et al. 2015

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Plant polyphenols represent a huge reservoir of bioactive compounds. Industrial chicory, an important crop from northwestern Europe, accumulates an original combination of such compounds, i.e., chlorogenic, isochlorogenic, caftaric, and chicoric acids arising from the phenylpropanoid pathway. For a complete understanding of these biochemical pathways, analyses of gene expression using quantitative real-time PCR (qRT-PCR) should be considered. Because cell cultures are a model of choice for specialized metabolism investigations, this study described for the first time the validation of reference genes for this system in chicory. Eighteen potential reference genes were obtained by mining expressed sequence tag databases of chicory for orthologs of Arabidopsis thaliana genes currently used as reference genes. Twelve genes passed the qRT-PCR standard requirements and their expression stability across different samples was tested using three distinct softwares: geNorm, NormFinder, and BestKeeper. In cell cultures grown under various conditions, TIP41 (TIP41 like protein) was shown to be the most stable gene. Further validation of the proposed reference genes was done by normalization of expression levels of a group of genes of interest. In order to assess the potentiality of the proposed list of candidate reference genes, theses genes were in parallel tested on another experimental design, i.e., chicory seedlings. In this case, the best reference gene identified was Clath (Clathrin adaptator complex subunit). The results highlight the importance of the use of properly validated reference genes to achieve relevant interpretation of qRT-PCR analyses. Here, we provide a list of reference genes suitable for future gene expression studies in chicory.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Selection and validation of reference genes for quantitative real-time PCR analysis of gene expression in Cichorium intybus

et al. 2015

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“…Glyceraldehyde-3-phosphate dehydrogenase is one of the reference genes commonly used to normalize RT-qPCR data. The use of GAPDH in many studies has provided good results (Cruz et al, 2009), otherwise, in other studies it is not recommended due to the variability of expression posed by different experimental conditions (Tong et al, 2009;Hu et al, 2014;Huang et al, 2014;Galli et al, 2015;Ye et al, 2015). In this paper, GAPDH has proven to be quite unstable in most of the algorithms used and also had a high CV%, 24.04 for 'Chimarrita/Aldrighi 1' and 38.53 for 'Chimarrita/Tsukuba 2' graft combination.…”

Section: Discussionmentioning

confidence: 88%

“…The reference genes used are generally those involved in basic cellular processes, such as primary metabolism and maintenance of cell structure (Expósito-Rodríguez et al, 2008). Those most commonly tested in RT-qPCR studies in plants include Actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GADPH), Elongation factor-1α (Ef-1α), Tubulin (α-TUA and β-TUA), ubiquitin (UBQ5, UBQ10), ribosomal RNA (18SrRNA, 40S), and cyclophilin (CYP) (Jain et al, 2006;Tong et al, 2009;Le et al, 2012;Huang et al, 2014;Hu et al, 2014;Niu et al, 2014;Moraes et al, 2015;Ye et al, 2015;Galli et al, 2015). Nevertheless, studies have shown that the expression of many of these reference genes may have different expression between genotypes, tissues, organs, and developmental stages (Tong et al, 2009;Le et al, 2012;Hu et al, 2014;Niu et al, 2014;Galli et al, 2015;Moraes et al, 2015;Ye et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Stable reference genes for studies of gene expression in Prunus persica under water stress

Rickes¹,

Klumb²,

Benitez³

et al. 2016

Aust J Crop Sci

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Strategies which allow the unequivocal identification of genes expressed in response to different treatments and/or stress conditions are important to obtain reliable results in plant physiology studies. However, in order to analyze the expression levels of target genes through RT-qPCR technique, it is essential to use model genes with uniform expression levels under specific experimental conditions. The present study aimed at investigating the stability of reference genes in peach leaves cultivar scion Chimarrita grafted onto 'Aldrighi 1' and 'Tsukuba 2' rootstock, subjected to water deficit for a 9-day period. The experiment was conducted in a completely randomized design with four treatments that correspond the evaluations period: zero (control), 4 th , 7 th , and 9 th stress day of water deficit. For each treatment three biological replicates was used. Eight reference genes were analyzed, among them: ACT, CYP2, Ef-1α, GAPDH, TUA, TUB, UBQ10 and 18SrRNA. For the normalization and validation of RT-qPCR data were used the four major software programs currently available: geNorm, NormFinder, BestKeeper, and Comparative ΔC T. The results showed that there was no influence of different plant grafting combinations in the expression of all reference genes evaluated, yet genes TUA and CYP2 had the most stable expression in the leaves of the peach plants under water deficit for 9-d, whereas genes Ef-1α and ACT were the least stable ones for the same stress conditions.

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“…As a result, use of these two pairs of co-regulated genes did not affect the final ranking of the reference genes by using geNorm software. Other methods such as NormFinder and BestKeeper, were reported to be less sensitive to co-regulation, and might serve as appropriate statistical applets to further assess the stability for reference gene expression (Huang et al 2014). In an effort to ensure the accuracy of the reference gene stability ranking and minimize bias introduced by the validation approach, four different statistical approaches, ∆Ct, geNorm, NormFinder, and BestKeeper, were used to identify the suitable reference genes for accurate normalization in this study.…”

Section: Discussionmentioning

confidence: 99%

Validation of reference genes for normalization of gene expression by qRT-PCR in a resveratrol-producing entophytic fungus (Alternaria sp. MG1)

Che

Shi

et al. 2016

AMB Expr

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Alternaria sp. MG1, an endophytic fungus isolated from Vitis vinifera, can independently produce resveratrol, indicating that this species contains the key genes for resveratrol biosynthesis. Identification of these key genes is essential to understand the resveratrol biosynthesis pathway in this strain, which is currently unknown in microorganisms. qRT-PCR is an efficient and widely used method to identify the key genes related to unknown pathways at the level of gene expression. Verification of stable reference genes in this strain is essential for qRT-PCR data normalization, although results have been reported for other Alternaria sp. strains. In this study, nine candidate reference genes including TUBA, EF1, EF2, UBC, UFD, RPS5, RPS24, ACTB and 18S were evaluated for expression stability in a diverse set of six samples representing different growth periods. We compared cell culture conditions and an optimized condition for resveratrol production. The comparison of the results was performed using four statistical softwares. A combination of TUBA and EF1 was found to be suitable for normalization of Alternaria sp. MG1 in different developmental stages, and 18S was found to be the least stable. The reference genes verified in this study will facilitate further research to explore gene expression and molecular mechanisms as well as the improvement of secondary metabolite yields in Alternaria sp. MG1. To our knowledge, this is the first validation of reference genes in Alternaria with the capability to produce resveratrol. Additionally, these results provide useful guidelines for the selection of reference genes in other Alternaria species.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0283-z) contains supplementary material, which is available to authorized users.

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Reference gene selection for quantitative real-time reverse-transcriptase PCR in orchardgrass subjected to various abiotic stresses

Cited by 41 publications

References 49 publications

Selection and validation of reference genes for quantitative real-time PCR analysis of gene expression in Cichorium intybus

Selection and validation of reference genes for quantitative real-time PCR analysis of gene expression in Cichorium intybus

Stable reference genes for studies of gene expression in Prunus persica under water stress

Validation of reference genes for normalization of gene expression by qRT-PCR in a resveratrol-producing entophytic fungus (Alternaria sp. MG1)

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