For Publishers
DiscoveryMetadataPeer reviewHostingPublishing
For Researchers
JoinPublishReviewCollect
Register
Blog
About
Search
Advanced search
My ScienceOpen
Search
For Publishers
For Researchers
Blog
About
137
views
53
references
 Top references
cited by
139
    
0 reviews
 Review
0
comments
 Comment
0
recommends
+1 Recommend
0 collections
    4
    shares
      238
      similar
       All similar
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions

      research-article

      Read this article at

      ScienceOpenPublisherPMC
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens ( Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress.

          Related collections

          Most cited references53

          • Record: found
          • Abstract: found
          • Article: not found

          Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress.

          Nathalie Nicot, Jean-Francois Hausman, Lucien Hoffmann … (2005)
          Plant stress studies are more and more based on gene expression. The analysis of gene expression requires sensitive, precise, and reproducible measurements for specific mRNA sequences. Real-time RT-PCR is at present the most sensitive method for the detection of low abundance mRNA. To avoid bias, real-time RT-PCR is referred to one or several internal control genes, which should not fluctuate during treatments. Here, the non-regulation of seven housekeeping genes (beta-tubulin, cyclophilin, actin, elongation factor 1-alpha (ef1alpha), 18S rRNA, adenine phosphoribosyl transferase (aprt), and cytoplasmic ribosomal protein L2) during biotic (late blight) and abiotic stresses (cold and salt stress) was tested on potato plants using geNorm software. Results from the three experimental conditions indicated that ef1alpha was the most stable among the seven tested. The expression of the other housekeeping genes tested varied upon stress. In parallel, a study of the variability of expression of hsp20.2, shown to be implicated in late blight stress, was realized. The relative quantification of the hsp20.2 gene varied according to the internal control and the number of internal controls used, thus highlighting the importance of the choice of internal controls in such experiments.
          Article 0 comments Cited 423 times – based on 0 reviews     Review now
            Bookmark
            • Record: found
            • Abstract: found
            • Article: found
            Is Open Access

            An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development

            Karen E Reid, Niclas Olsson, James Schlosser … (2006)
            Background Accuracy in quantitative real-time RT-PCR is dependent on high quality RNA, consistent cDNA synthesis, and validated stable reference genes for data normalization. Reference genes used for normalization impact the results generated from expression studies and, hence, should be evaluated prior to use across samples and treatments. Few statistically validated reference genes have been reported in grapevine. Moreover, success in isolating high quality RNA from grapevine tissues is typically limiting due to low pH, and high polyphenolic and polysaccharide contents. Results We describe optimization of an RNA isolation procedure that compensates for the low pH found in grape berries and improves the ability of the RNA to precipitate. This procedure was tested on pericarp and seed developmental series, as well as steady-state leaf, root, and flower tissues. Additionally, the expression stability of actin, AP47 (clathrin-associated protein), cyclophilin, EF1-α (elongation factor 1-α), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), MDH (malate dehydrogenase), PP2A (protein phosphatase), SAND, TIP41, α-tubulin, β-tubulin, UBC (ubiquitin conjugating enzyme), UBQ-L40 (ubiquitin L40) and UBQ10 (polyubiquitin) were evaluated on Vitis vinifera cv. Cabernet Sauvignon pericarp using three different statistical approaches. Although several of the genes proved to be relatively stable, no single gene outperformed all other genes in each of the three evaluation methods tested. Furthermore, the effect of using one reference gene versus normalizing to the geometric mean of several genes is presented for the expression of an aquaporin and a sucrose transporter over a developmental series. Conclusion In order to quantify relative transcript abundances accurately using real-time RT-PCR, we recommend that combinations of several genes be used for normalization in grape berry development studies. Our data support GAPDH, actin, EF1-α and SAND as the most relevant reference genes for this purpose.
            Article 0 comments Cited 286 times – based on 0 reviews     Review now
              Bookmark
              • Record: found
              • Abstract: found
              • Article: found
              Is Open Access

              Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

              Marino Expósito-Rodríguez, Andres A Bazan-Borges, Andrés Borges-Pérez … (2008)
              Background The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR. Results In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC), SGN-U321250 (TIP41), SGN-U346908 ("Expressed") and SGN-U316474 (SAND) genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process. Conclusion This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time RT-PCR studies of gene expression during tomato development process. From our study a tool-kit of control genes emerges that outperform the traditional genes in terms of expression stability.
              Article 0 comments Cited 261 times – based on 0 reviews     Review now
                Bookmark
                All references

                Author and article information

                Contributors
                : Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, USA )
                ISSN (Electronic): 1932-6203
                Publication date Collection: 2012
                Publication date (Electronic): 9 February 2012
                Volume: 7
                Issue: 2
                Electronic Location Identifier: e31263
                Affiliations
                [1 ]Laboratório de Biotecnologia, Centro de Citricultura Sylvio Moreira, Cordeirópolis-São Paulo, Brazil
                [2 ]Instituto de Biologia, Universidade Estadual de Campinas, Campinas-São Paulo, Brazil
                [3 ]Laboratório de Genética Molecular Vegetal, Departamento de Genética, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
                [4 ]Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
                East Carolina University, United States of America
                Author notes

                Conceived and designed the experiments: VM MAF MAM. Analyzed the data: VM MAF MRA. Wrote the paper: VM. Read, corrected and approved the final manuscript: VM KSK MAF MRA RMS LB CMR MAM. Performed the developmental stage assay: VM. Performed the biotic assays: KSK RMS LB CMR. Carried out the RT-qPCR experiments (primer design, optimizations, acquisitions, analysis and interpretation of data): VM KSK. Coordinated the study: MAM.

                Article
                Publisher ID: PONE-D-11-17696
                DOI: 10.1371/journal.pone.0031263
                PMC ID: 3276578
                PubMed ID: 22347455
                SO-VID: a793908c-4a47-4fee-ad92-aebb8c47cebe
                Copyright © Mafra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                Date received : 8 September 2011
                Date accepted : 4 January 2012
                Page count
                Pages: 11
                Categories
                Subject: Research Article
                Subject: Agriculture
                Subject: Crops
                Subject: Biology
                Subject: Genetics
                Subject: Plant Genetics
                Subject: Genomics
                Subject: Plant Science

                ScienceOpen disciplines: Uncategorized
                Data availability:
                ScienceOpen disciplines: Uncategorized

                Comments

                Comment on this article

                scite_

                Similar content238

                See all similar

                Cited by137

                See all cited by

                Most referenced authors877

                See all reference authors