Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions

Die, José V.; Román, Belén; Nadal, Salvador; González-Verdejo, Clara Isabel

doi:10.1007/s00425-010-1158-1

Cited by 168 publications

(131 citation statements)

References 38 publications

Supporting

Mentioning

119

Contrasting

Unclassified

Order By: Relevance

“…These results suggested that reference genes validated for a single genotype may not be the most appropriate reference gene for use when comparing gene expression across diverse cultivars. Multiple candidate reference genes for use with pea have only been validated against a single yellow animal feed cultivar, Athos [6], although the 18s rRNA gene has also been used as a reference gene to examine the yellow animal feed pea cultivar Alaska [14]. In this study, we examined several different candidate reference genes among three food grade spring green cultivars and two animal feed spring yellow cultivars.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Expression Stability of Candidate References Genes among Green and Yellow Pea Cultivars (<i>Pisum sativum</i> L.) Subjected to Abiotic and Biotic Stress

Saha¹,

Vandemark²

2012

AJPS

View full text Add to dashboard Cite

Dry pea (Pisum sativum L.) is grown as human and animal feed throughout the world. Large yield losses in pea due to biotic and abiotic stresses compel an improved understanding of mechanisms of stress tolerance and genetic determinants conditioning these tolerances. The availability of stably expressed reference genes is a prerequisite for examining differential gene expression. The objective of this study was to examine the expression profile of several candidate reference genes across a broad range of commercial pea cultivars. Expression profiles of five candidate reference genes; 18s rRNA, actin, TIF, β tubulin-2 and β tubulin-3 were examined. Relative quantifications of candidate reference genes were estimated from control plants, plants after 48 h of cold treatment, and plants 24 and 48 h after inoculation with Sclerotinia sclerotiorum, the causal agent of white mold disease of pea. RT-qPCR was performed on cDNA synthesized from three food grade spring peas, Ariel, Aragorn, and Sterling, and two spring yellow peas, Delta and Universal, which are used as animal feed. Analysis of variance (ANOVA) of C T values demonstrated significant variation between varieties and treatments under cold and disease conditions. The most abundant transcripts among tested reference genes were for 18s rRNA. Stability analysis indicated that TIF and β tubulin-3 genes were the most stably expressed candidate genes under both cold and disease stress and could serve as reference genes across a wide range of pea cultivars.

show abstract

Section: Discussionmentioning

confidence: 99%

Evaluation of Expression Stability of Candidate References Genes among Green and Yellow Pea Cultivars (<i>Pisum sativum</i> L.) Subjected to Abiotic and Biotic Stress

Saha¹,

Vandemark²

2012

AJPS

View full text Add to dashboard Cite

show abstract

“…The commonly used housekeeping genes were selected as candidate reference genes according to the previous reports in other plants (Reid et al 2006;Hong et al 2008;ExpositoRodriguez et al 2008;Kumeta and Ito 2010;Nicot et al 2005;Die et al 2010;Chen et al 2011). Ortholog sequences of these genes in A. sinensis were obtained in the 454 highthroughput sequencing library and identified through BLASTX against GenBank.…”

Section: Candidate Reference Gene Selection and Primer Designmentioning

confidence: 99%

Selection and validation of reference genes for studying stress-related agarwood formation of Aquilaria sinensis

et al. 2012

View full text Add to dashboard Cite

show abstract

“…Northern blotting, semi-quantitative reverse transcription-PCR, and reverse transcription quantitative real-time PCR (RT-qPCR, Bustin et al 2009) have been frequently used, and RT-qPCR is the best method available for determining changes in gene expression, due to its higher sensitivity, specificity, and broad quantification range of up to seven orders of magnitude (reviewed in Bustin 2002;Wong and Medrano 2005;Artico et al 2010). Therefore, RT-qPCR has become the preferred method for the validation of high-throughput or microarray results and the quantitation of gene expression (Chuaqui et al 2002;Czechowski et al 2005;Die et al 2010).…”

Section: Introductionmentioning

confidence: 99%

“…Although RT-qPCR is widely used to quantify biologically relevant changes in mRNA levels, the accuracy of RT-qPCR is influenced by a number of variables, including the variability in RNA samples, extraction protocols (particularly due to the co-purification of inhibitors), and efficiencies of the RT and PCR (Nolan et al 2006;Die et al 2010;Schmidt and Delaney 2010). Consequently, a normalization step is an essential pre-requisite.…”

Section: Introductionmentioning

confidence: 99%

Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions

Chen

Zhong

Kuang

et al. 2011

Planta

339

244

View full text Add to dashboard Cite

Reverse transcription quantitative real-time PCR (RT-qPCR) is a sensitive technique for quantifying gene expression, but its success depends on the stability of the reference gene(s) used for data normalization. Only a few studies on validation of reference genes have been conducted in fruit trees and none in banana yet. In the present work, 20 candidate reference genes were selected, and their expression stability in 144 banana samples were evaluated and analyzed using two algorithms, geNorm and NormFinder. The samples consisted of eight sample sets collected under different experimental conditions, including various tissues, developmental stages, postharvest ripening, stresses (chilling, high temperature, and pathogen), and hormone treatments. Our results showed that different suitable reference gene(s) or combination of reference genes for normalization should be selected depending on the experimental conditions. The RPS2 and UBQ2 genes were validated as the most suitable reference genes across all tested samples. More importantly, our data further showed that the widely used reference genes, ACT and GAPDH, were not the most suitable reference genes in many banana sample sets. In addition, the expression of MaEBF1, a gene of interest that plays an important role in regulating fruit ripening, under different experimental conditions was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene(s) selection under different experimental conditions and a foundation for more accurate and widespread use of RT-qPCR in banana.

show abstract

Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions

Cited by 168 publications

References 38 publications

Evaluation of Expression Stability of Candidate References Genes among Green and Yellow Pea Cultivars (<i>Pisum sativum</i> L.) Subjected to Abiotic and Biotic Stress

Evaluation of Expression Stability of Candidate References Genes among Green and Yellow Pea Cultivars (<i>Pisum sativum</i> L.) Subjected to Abiotic and Biotic Stress

Selection and validation of reference genes for studying stress-related agarwood formation of Aquilaria sinensis

Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions

Contact Info

Product

Resources

About

Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions

Cited by 168 publications

References 38 publications

Evaluation of Expression Stability of Candidate References Genes among Green and Yellow Pea Cultivars (&lt;i&gt;Pisum sativum&lt;/i&gt; L.) Subjected to Abiotic and Biotic Stress

Evaluation of Expression Stability of Candidate References Genes among Green and Yellow Pea Cultivars (&lt;i&gt;Pisum sativum&lt;/i&gt; L.) Subjected to Abiotic and Biotic Stress

Selection and validation of reference genes for studying stress-related agarwood formation of Aquilaria sinensis

Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions

Contact Info

Product

Resources

About

Evaluation of Expression Stability of Candidate References Genes among Green and Yellow Pea Cultivars (<i>Pisum sativum</i> L.) Subjected to Abiotic and Biotic Stress

Evaluation of Expression Stability of Candidate References Genes among Green and Yellow Pea Cultivars (<i>Pisum sativum</i> L.) Subjected to Abiotic and Biotic Stress