Selection of Reference Genes for Transcriptional Analysis of Edible Tubers of Potato (Solanum tuberosum L.)

Mariot, Roberta Fogliatto; Oliveira, Luisa Abruzzi de; Voorhuijzen, Marleen M.; Staats, Martijn; Hutten, R.C.B.; Dijk, Jeroen P. van; Kok, E.J.; Frazzon, Jeverson

doi:10.1371/journal.pone.0120854

Cited by 23 publications

(22 citation statements)

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“…Primer pairs were designed from these sequences with the NCBI Primer-BLAST program (Table 1 ) ( http://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) [ 18 ]. Primers were designed across exon boundaries to avoid genomic DNA contamination and exon analysis using Splign ( http://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi ) [ 11 ]. Before RT-qPCR analysis, PCR was performed using primers as shown in Table 1 to determine the size specificity of the amplicons, then electrophoresed on ethoxylated gels and ethidium bromide, and the target amplicons were sequenced to confirm the identity of the PCR product.…”

Section: Methodsmentioning

confidence: 99%

“…In potato, EF1 - α was confirmed to be the most suitable reference gene under salt stress and late blight, and EF1 - α and APRT were considered to be the most stable under cold stress [ 9 , 10 ]. In addition, sec3 , CUL3A , and C2 were found to be most suitable for screening of reference genes in edible tubers [ 11 ]. However, the stability of these reference genes under other abiotic stresses has not been confirmed.…”

Section: Introductionmentioning

confidence: 99%

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Selection and validation of reference genes for RT-qPCR analysis in potato under abiotic stress

et al. 2017

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BackgroundReal-time quantitative PCR (RT-qPCR) is the most commonly used method for accurately detecting gene expression patterns. As part of RT-qPCR analysis, normalization of the data requires internal control gene(s) that display uniform expression under different biological conditions. However, no invariable internal control gene exists, and therefore more than one reference gene is needed to normalize RT-qPCR results. Identification of stable reference genes in potato will improve assay accuracy for selecting stress-tolerance genes and identifying molecular mechanisms conferring stress tolerance in this species.ResultsIn the experiment, we assessed the expression of eight candidate internal control genes, namely elongation factor-1alpha (EF1α), actin, tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), adenine phosphoribosyl transferase (APRT), 60S ribosomal protein L8 (L8), Cullin 3A (CUL3A), and exocyst complex component sec3 (sec3), in a diverse set of potato samples representing drought stress and osmotic stress challenges, and using geNorm, NormFinder, BestKeeper and RefFinder softwares.ConclusionsThe results indicated that EF1α and sec3 were the most stably expressed genes in the potato under drought and osmotic stress conditions. This work will facilitate future work on gene expression studies in potato and also benefit other species of the Solanaceae, such as tomato.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Selection and validation of reference genes for RT-qPCR analysis in potato under abiotic stress

et al. 2017

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“…Various statistical tools, e.g., geNorm, NormFinder, BestKeeper and RefFinder, have been developed to determine reference gene suitability for qPCR data normalization (Vandesompele et al, 2002 ; Andersen et al, 2004 ; Pfaffl et al, 2004 ). Stable reference gene validation has been performed in many plant species, including model species and crop plants such as Arabidopsis thaliana (Czechowski et al, 2005 ; Remans et al, 2008 ), Brassica napus (Yang et al, 2014a ; Machado et al, 2015 ), Hordium vulgare (Zmienko et al, 2015 ), Fagopyrum tataricum (Demidenko et al, 2011 ), Cajanus cajan (Sinha et al, 2015 ), Oryza sativum (Jain et al, 2006 ; Li et al, 2010 ; Ji et al, 2014 ), Arachis hypogaea (Reddy et al, 2013 ), Pennisetum glaucum (Saha and Blumwald, 2014 ; Reddy et al, 2015b ), Solanum tuberosum (Nicot et al, 2005 ; Mariot et al, 2015 ), Glycine max (Jian et al, 2008 ; Nakayama et al, 2014 ), Saccharum officinarum (Iskandar et al, 2004 ; Ling et al, 2015 ), Lycopersicon esculentum (Expósito-Rodríguez et al, 2008 ), Triticum aestivum (Paolacci et al, 2009 ), and Zea mays (Manoli et al, 2012 ; Lin et al, 2014 ).…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Sorghum [Sorghum bicolor (L.)] Reference Genes in Various Tissues and under Abiotic Stress Conditions for Quantitative Real-Time PCR Data Normalization

et al. 2016

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Accurate and reliable gene expression data from qPCR depends on stable reference gene expression for potential gene functional analyses. In this study, 15 reference genes were selected and analyzed in various sample sets including abiotic stress treatments (salt, cold, water stress, heat, and abscisic acid) and tissues (leaves, roots, seedlings, panicle, and mature seeds). Statistical tools, including geNorm, NormFinder and RefFinder, were utilized to assess the suitability of reference genes based on their stability rankings for various sample groups. For abiotic stress, PP2A and CYP were identified as the most stable genes. In contrast, EIF4α was the most stable in the tissue sample set, followed by PP2A; PP2A was the most stable in all the sample set, followed by EIF4α. GAPDH, and UBC1 were the least stably expressed in the tissue and all the sample sets. These results also indicated that the use of two candidate reference genes would be sufficient for the optimization of normalization studies. To further verify the suitability of these genes for use as reference genes, SbHSF5 and SbHSF13 gene expression levels were normalized using the most and least stable sorghum reference genes in root and water stressed-leaf tissues of five sorghum varieties. This is the first systematic study of the selection of the most stable reference genes for qPCR-related assays in Sorghum bicolor that will potentially benefit future gene expression studies in sorghum and other closely related species.

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“…1 and 2). An idea for exploring a reference gene in such species is to test genes orthologous to a reference gene used in other species, as previously conducted in several plant species including grapevine, tomato and potato (Reid et al, 2006;Dekkers et al, 2012;Mariot et al, 2015). To briefly check if this might work with our candidates, αcp and other CK2A genes were explored and evaluated as reference genes in the four excluded species.…”

Section: Discussionmentioning

confidence: 99%

Identification of reference genes for quantitative expression analysis using large-scale RNA-seq data of <i>Arabidopsis thaliana</i> and model crop plants

et al. 2016

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In quantitative gene expression analysis, normalization using a reference gene as an internal control is frequently performed for appropriate interpretation of the results. Efforts have been devoted to exploring superior novel reference genes using microarray transcriptomic data and to evaluating commonly used reference genes by targeting analysis. However, because the number of specifically detectable genes is totally dependent on probe design in the microarray analysis, exploration using microarray data may miss some of the best choices for the reference genes. Recently emerging RNA sequencing (RNA-seq) provides an ideal resource for comprehensive exploration of reference genes since this method is capable of detecting all expressed genes, in principle including even unknown genes. We report the results of a comprehensive exploration of reference genes using public RNA-seq data from plants such as Arabidopsis thaliana (Arabidopsis), Glycine max (soybean), Solanum lycopersicum (tomato) and Oryza sativa (rice). To select reference genes suitable for the broadest experimental conditions possible, candidates were surveyed by the following four steps: (1) evaluation of the basal expression level of each gene in each experiment; (2) evaluation of the expression stability of each gene in each experiment; (3) evaluation of the expression stability of each gene across the experiments; and (4) selection of top-ranked genes, after ranking according to the number of experiments in which the gene was expressed stably. Employing this procedure, 13, 10, 12 and 21 top candidates for reference genes were proposed in Arabidopsis, soybean, tomato and rice, respectively. Microarray expression data confirmed that the expression of the proposed reference genes under broad experimental conditions was more stable than that of commonly used reference genes. These novel reference genes will be useful for analyzing gene expression profiles across experiments carried out under various experimental conditions.

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Selection of Reference Genes for Transcriptional Analysis of Edible Tubers of Potato (Solanum tuberosum L.)

Cited by 23 publications

References 50 publications

Selection and validation of reference genes for RT-qPCR analysis in potato under abiotic stress

Selection and validation of reference genes for RT-qPCR analysis in potato under abiotic stress

Evaluation of Sorghum [Sorghum bicolor (L.)] Reference Genes in Various Tissues and under Abiotic Stress Conditions for Quantitative Real-Time PCR Data Normalization

Identification of reference genes for quantitative expression analysis using large-scale RNA-seq data of <i>Arabidopsis thaliana</i> and model crop plants

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