Reference gene selection for quantitative real-time polymerase chain reaction in Populus

Xu, Meng; Zhang, Bo; Su, Xiaohua; Zhang, Shougong; Huang, Minren

doi:10.1016/j.ab.2010.08.044

Cited by 126 publications

(104 citation statements)

References 16 publications

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“…Greater expression stability has already been demonstrated for the plant genes ELF1B (Jian et al, 2008;Hu et al, 2009), ACTB (Stolf-Moreira et al, 2011), and TUA (Jian et al, 2008;de Almeida et al, 2010). Previous plant gene expression studies have classified the 18S gene among the most stable (Stolf-Moreira et al, 2011;Xu et al, 2011), whereas in other studies, it is ranked as the least stable (de Almeida et al, 2010), which is consistent with our findings.…”

Section: Discussionsupporting

confidence: 82%

“…Compared with other studies of expression stability, such as adventitious rooting in Eucalyptus globulus (de Almeida et al, 2010) and Populus (Xu et al, 2011), in the different soybean tissues (Jian et al, 2008;Hu et al, 2009) and drought-stressed soybean roots and leaves (Stolf-Moreira et al, 2011), 18S transcripts were the most abundant (de Almeida et al, 2010;Stolf-Moreira et al, 2011). TUA2 [called TUB by Jian et al (2008)] and ELF1B exhibited the largest (Jian et al, 2008) and smallest (Hu et al, 2009) variations in Ct values, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…Although these reference genes require stable expression in general, they can nonetheless vary across developmental stages, tissues, or experimental treatments (Jian et al, 2008;Hu et al, 2009). Therefore, to avoid biasing the normalized expression of the target gene because of gene-specific variation in the reference gene, it is necessary to validate the best candidate reference genes (CRGs) for each experimental treatment (Xu et al, 2011). To our knowledge, this study is the first to evaluate the expression stability of CRGs in root tissues from hypoxia-stressed soybean plants.…”

Section: Introductionmentioning

confidence: 99%

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Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions

et al. 2014

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ABSTRACT. Quantitative real-time polymerase chain reaction (RTqPCR) is a powerful tool used to measure gene expression. However, because of its high sensitivity, the method is strongly influenced by the quality and concentration of the template cDNA and by the amplification efficiency. Relative quantification is an effective strategy for correcting random and systematic errors by using the expression level of reference gene(s) to normalize the expression level of the genes of interest. To identify soybean reference genes for use in studies of flooding stress, we compared 5 candidate reference genes (CRGs) with the NormFinder and GeNorm programs to select the best internal control. The expression Gene expression in flooding-stressed soybeans stability of the CRGs was evaluated in root tissues from soybean plants subjected to hypoxic conditions. Elongation factor 1-beta and actin-11 were identified as the most appropriate genes for RT-qPCR normalization by both the NormFinder and GeNorm analyses. The expression profiles of the genes for alcohol dehydrogenase 1, sucrose synthase 4, and ascorbate peroxidase 2 were analyzed by comparing different normalizing combinations (including no normalization) of the selected reference genes. Here, we have identified potential genes for use as references for RT-qPCR normalization in experiments with soybean roots growing in O 2 -depleted environments, such as flooding-stressed plants.

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Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions

et al. 2014

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“…However, several studies have demonstrated that the expression levels of many so-called reference genes, such as the 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), b-Actin, cyclophilin (Cyp), elongation factor-1-A (EF1A) and the 60S ribosomal protein L2 (rpl2) may differ greatly among different tissue types, species or in response to various stresses (Czechowski 2005;Migocka and Papierniak 2010;Xu et al 2011). Recognizing the importance of reference genes in normalization of qPCR data, valuation of suitable reference genes has been conducted in various plants, such as Arabidopsis thaliana (Remans et al 2008), Cucumis sativus (Migocka and Papierniak 2010), and Populous (Xu et al 2011), under specific conditions. Hitherto suitable internal controls for gene expression studies have not been defined for mangroves.…”

Section: Introductionmentioning

confidence: 99%

Identification of suitable reference genes in mangrove Aegiceras corniculatum under abiotic stresses

Yang

Wang

2015

Ecotoxicology

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Gene expression studies could provide insight into the physiological mechanisms and strategies used by plants under stress conditions. Selection of suitable internal control gene(s) is essential to accurately assess gene expression levels. For the mangrove plant, Aegiceras corniculatum, reliable reference genes to normalize real-time quantitative PCR data have not been previously investigated. In this study, the expression stabilities of five candidate reference genes [glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 18SrRNA, β-Actin, 60S ribosomal protein L2, and elongation factor-1-A] were determined in leaves of A. corniculatum treated by cold, drought, salt, heavy metals, and pyrene and in different tissues of A. corniculatum under normal condition. Two software programs (geNorm and NormFinder) were employed to analyze and rank the tested genes. Results showed that GAPDH was the most suitable reference gene in A. corniculatum and the combination of two or three genes was recommended for greater accuracy. To assess the value of these tested genes as internal controls, the relative quantifications of CuZnSOD gene were also conducted. Results showed that the relative expression levels of CuZnSOD gene varied depending on the internal reference genes used, which highlights the importance of the choice of suitable internal controls in gene expression studies. Furthermore, the results also confirmed that GAPDH was a suitable reference gene for qPCR normalization in A. corniculatum under abiotic stresses. Identification of A. corniculatum reference gens in a wide range of experimental samples will provide a useful reference in future gene expression studies in this species, particularly involving similar stresses.

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“…Moreover, all genes involved in the same experiment should have similar ranges of expression, to become possible of being analyzed (Cappelli et al, 2008). Reference genes are constitutively and universally expressed, and their products are necessary for cytoarchitecture or basic metabolism (Bustin, 2002;Xu et al, 2011). However, traditional reference genes, such as Act, Tubulin, EF1-α, and GAPDH, are not always universally expressed in different cases (Glare et al, 2002;Everaert et al, 2011;Migocka and Papierniak, 2011).…”

Section: Introductionmentioning

confidence: 99%

Identification of reference genes for RT-qPCR analysis in peach genotypes with contrasting chilling requirements

et al. 2017

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ABSTRACT. Selecting and validating reference genes are the first steps in studying gene expression by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). The present study aimed to evaluate the stability of five reference genes for the purpose of normalization when studying gene expression in various cultivars of Prunus persica with different chilling requirements. Flower bud tissues of nine peach genotypes from Embrapa's peach breeding program with different chilling requirements were used, and five candidate reference genes based on the RT-qPCR that were useful for studying the relative quantitative gene expression and stability were evaluated using geNorm, NormFinder, and bestKeeper software packages. The results indicated that among the genes tested, the most stable genes to be used as reference genes are Act and UBQ10. This study is the first survey of the stability of reference genes in peaches under chilling stress and provides guidelines for more accurate RT-qPCR results.

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Reference gene selection for quantitative real-time polymerase chain reaction in Populus

Cited by 126 publications

References 16 publications

Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions

Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions

Identification of suitable reference genes in mangrove Aegiceras corniculatum under abiotic stresses

Identification of reference genes for RT-qPCR analysis in peach genotypes with contrasting chilling requirements

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