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      Assessing Reference Genes for Accurate Transcript Normalization Using Quantitative Real-Time PCR in Pearl Millet [ Pennisetum glaucum (L.) R. Br.]

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          Abstract

          Pearl millet [ Pennisetum glaucum (L.) R.Br.], a close relative of Panicoideae food crops and bioenergy grasses, offers an ideal system to perform functional genomics studies related to C4 photosynthesis and abiotic stress tolerance. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) provides a sensitive platform to conduct such gene expression analyses. However, the lack of suitable internal control reference genes for accurate transcript normalization during qRT-PCR analysis in pearl millet is the major limitation. Here, we conducted a comprehensive assessment of 18 reference genes on 234 samples which included an array of different developmental tissues, hormone treatments and abiotic stress conditions from three genotypes to determine appropriate reference genes for accurate normalization of qRT-PCR data. Analyses of Ct values using Stability Index, BestKeeper, ΔCt, Normfinder, geNorm and RefFinder programs ranked PP2A, TIP41, UBC2, UBQ5 and ACT as the most reliable reference genes for accurate transcript normalization under different experimental conditions. Furthermore, we validated the specificity of these genes for precise quantification of relative gene expression and provided evidence that a combination of the best reference genes are required to obtain optimal expression patterns for both endogeneous genes as well as transgenes in pearl millet.

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          Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

          A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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            Primer3Plus, an enhanced web interface to Primer3

            Here we present Primer3Plus, a new web interface to the popular Primer3 primer design program as an enhanced alternative for the CGI- scripts that come with Primer3. Primer3 consists of a command line program and a web interface. The web interface is one large form showing all of the possible options. This makes the interface powerful, but at the same time confusing for occasional users. Primer3Plus provides an intuitive user interface using present-day web technologies and has been developed in close collaboration with molecular biologists and technicians regularly designing primers. It focuses on the task at hand, and hides detailed settings from the user until these are needed. We also added functionality to automate specific tasks like designing primers for cloning or step-wise sequencing. Settings and designed primer sequences can be stored locally for later use. Primer3Plus supports a range of common sequence formats, such as FASTA. Finally, primers selected by Primer3Plus can be sent to an order form, allowing tight integration into laboratory ordering systems. Moreover, the open architecture of Primer3Plus allows easy expansion or integration of external software packages. The Primer3Plus Perl source code is available under GPL license from SourceForge. Primer3Plus is available at http://www.bioinformatics.nl/primer3plus.
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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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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                29 August 2014
                : 9
                : 8
                : e106308
                Affiliations
                [1]Department of Plant Sciences, University of California Davis, Davis, California, United States of America
                National Key Laboratory of Crop Genetic Improvement, China
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: PS EB. Performed the experiments: PS. Analyzed the data: PS EB. Contributed reagents/materials/analysis tools: EB. Contributed to the writing of the manuscript: PS EB.

                Article
                PONE-D-14-22690
                10.1371/journal.pone.0106308
                4149553
                25170776
                9e7e797b-380e-4079-b5c9-67745b1ec6e0
                Copyright @ 2014

                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
                : 23 May 2014
                : 4 August 2014
                Page count
                Pages: 16
                Funding
                This work is funded by The United States Agency for International Development (USAID) under the Grant No. APS M/OAA/GRO/EGAS-11-002011. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biotechnology
                Plant Biotechnology
                Plant Genomics
                Plant Science
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

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