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      Selection of reference genes suitable for normalization of qPCR data under abiotic stresses in bioenergy crop Arundo donax L.

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      Scientific Reports
      Nature Publishing Group UK

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          Abstract

          Suitable reference gene selection in qRT-PCR is a key pre-requisite to produce reliable data in gene expression analyses. In this study, novel primers for six commonly used reference genes ( AC1, TLF, Act2, TUB α, EF-1α and GAPDH) plus two new candidates ( pDUF221 and RPN6) were designed and comparatively tested for expression stability under abiotic stresses (osmotic, heavy metal and heat shock) in shoot, root and their combination of Arundo donax L., a raising non-food energy crop. Expression stability rankings from the most to the least stable gene in each condition and in two tissues (young shoots and roots) were generated with geNorm, NormFinder and BestKeeper programs. All programs provided similar rankings and, strikingly, in most cases identified one of the new candidates, RPN6, as the most suitable reference gene. This novel set of reliable references allows to choose either the best combination of reference genes across multiple stress/organ conditions or to select condition-specific genes that can improve the quality of qRT-PCR analysis. This work provides a solid basis for the functional characterization of A. donax, by enabling accurate quantification of the transcriptional responsiveness under a series of common stress conditions of any gene of interest in this promising biomass/bioenergy species.

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          Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR

          Background Control genes, which are often referred to as housekeeping genes, are frequently used to normalise mRNA levels between different samples. However, the expression level of these genes may vary among tissues or cells and may change under certain circumstances. Thus, the selection of housekeeping genes is critical for gene expression studies. To address this issue, 7 candidate housekeeping genes including several commonly used ones were investigated in isolated human reticulocytes. For this, a simple ΔCt approach was employed by comparing relative expression of 'pairs of genes' within each sample. On this basis, stability of the candidate housekeeping genes was ranked according to repeatability of the gene expression differences among 31 samples. Results Initial screening of the expression pattern demonstrated that 1 of the 7 genes was expressed at very low levels in reticulocytes and was excluded from further analysis. The range of expression stability of the other 6 genes was (from most stable to least stable): GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L (HBS1-like protein) and AHSP (alpha haemoglobin stabilising protein), followed by B2M (beta-2-microglobulin). Conclusion Using this simple approach, GAPDH was found to be the most suitable housekeeping gene for expression studies in reticulocytes while the commonly used B2M should be avoided.
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            Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought-responsive gene expression.

            Transcription factors DREB1A/CBF3 and DREB2A specifically interact with cis-acting dehydration-responsive element/C-repeat (DRE/CRT) involved in cold and drought stress-responsive gene expression in Arabidopsis thaliana. Intact DREB2A expression does not activate downstream genes under normal growth conditions, suggesting that DREB2A requires posttranslational modification for activation, but the activation mechanism has not been clarified. DREB2A domain analysis using Arabidopsis protoplasts identified a transcriptional activation domain between residues 254 and 335, and deletion of a region between residues 136 and 165 transforms DREB2A to a constitutive active form. Overexpression of constitutive active DREB2A resulted in significant drought stress tolerance but only slight freezing tolerance in transgenic Arabidopsis plants. Microarray and RNA gel blot analyses revealed that DREB2A regulates expression of many water stress-inducible genes. However, some genes downstream of DREB2A are not downstream of DREB1A, which also recognizes DRE/CRT but functions in cold stress-responsive gene expression. Synthetic green fluorescent protein gave a strong signal in the nucleus under unstressed control conditions when fused to constitutive active DREB2A but only a weak signal when fused to full-length DREB2A. The region between DREB2A residues 136 and 165 plays a role in the stability of this protein in the nucleus, which is important for protein activation.
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              Reference genes in real-time PCR

              This paper aims to discuss various aspects of the use of reference genes in qPCR technique used in the thousands of present studies. Most frequently, these are housekeeping genes and they must meet several criteria so that they can lay claim to the name. Lots of papers report that in different conditions, for different organisms and even tissues the basic assumption—the constant level of the expression is not maintained for many genes that seem to be perfect candidates. Moreover, their transcription can not be affected by experimental factors. Sounds simple and clear but a great number of designed protocols and lack of consistency among them brings confusion on how to perform experiment properly. Since during selection of the most stable normalizing gene we can not use any reference gene, different ways and algorithms for their selection were developed. Such methods, including examples of best normalizing genes in some specific cases and possible mistakes are presented based on available sources. Numerous examples of reference genes applications, which are usually in too few numbers in relevant articles not allowing to make a solid fundament for a reader, will be shown along with instructive compilations to make an evidence for presented statements and an arrangement of future qPCR experiments. To include all the pitfalls and problems associated with the normalization methods there is no way not to begin from sample preparation and its storage going through candidate gene selection, primer design and statistical analysis. This is important because numerous short reviews available cover the topic only in lesser extent at the same time giving the reader false conviction of complete topic recognition.
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                Author and article information

                Contributors
                mingai.li@fmach.it
                claudio.varotto@fmach.it
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                6 September 2017
                6 September 2017
                2017
                : 7
                : 10719
                Affiliations
                [1 ]ISNI 0000 0004 1755 6224, GRID grid.424414.3, Department of Biodiversity and Molecular Ecology, , Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, ; 38010 S. Michele all’Adige (TN), Italy
                [2 ]ISNI 0000 0004 1757 1758, GRID grid.6292.f, Department of Agricultural Sciences, , University of Bologna, ; Bologna, Italy
                Article
                11019
                10.1038/s41598-017-11019-0
                5587670
                28878356
                e3654b9e-a011-4b80-b9e3-41cc77a2e18f
                © The Author(s) 2017

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 22 May 2017
                : 16 August 2017
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