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      The need for transparency and good practices in the qPCR literature

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      Nature Methods
      Springer Science and Business Media LLC

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          Abstract

          Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

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          Most cited references18

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          The real-time polymerase chain reaction.

          The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year's Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413-417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.
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            How to do successful gene expression analysis using real-time PCR.

            Reverse transcription quantitative PCR (RT-qPCR) is considered today as the gold standard for accurate, sensitive and fast measurement of gene expression. Unfortunately, what many users fail to appreciate is that numerous critical issues in the workflow need to be addressed before biologically meaningful and trustworthy conclusions can be drawn. Here, we review the entire workflow from the planning and preparation phase, over the actual real-time PCR cycling experiments to data-analysis and reporting steps. This process can be captured with the appropriate acronym PCR: plan/prepare, cycle and report. The key message is that quality assurance and quality control are essential throughout the entire RT-qPCR workflow; from living cells, over extraction of nucleic acids, storage, various enzymatic steps such as DNase treatment, reverse transcription and PCR amplification, to data-analysis and finally reporting. Copyright 2009 Elsevier Inc. All rights reserved.
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              Is Open Access

              MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments

              The conclusions of thousands of peer-reviewed publications rely on data obtained using fluorescence-based quantitative real-time PCR technology. However, the inadequate reporting of experimental detail, combined with the frequent use of flawed protocols is leading to the publication of papers that may not be technically appropriate. We take the view that this problem requires the delineation of a more transparent and comprehensive reporting policy from scientific journals. This editorial aims to provide practical guidance for the incorporation of absolute minimum standards encompassing the key assay parameters for accurate design, documentation and reporting of qPCR experiments (MIQE précis) and guidance on the publication of pure 'reference gene' articles.
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                Author and article information

                Journal
                Nature Methods
                Nat Methods
                Springer Science and Business Media LLC
                1548-7091
                1548-7105
                November 2013
                October 30 2013
                November 2013
                : 10
                : 11
                : 1063-1067
                Article
                10.1038/nmeth.2697
                24173381
                44327f87-1a08-4589-b2f7-484d127de647
                © 2013

                http://www.springer.com/tdm

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