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Aug 1, 2009 (Vol. 29, No. 14)

PCR Technology Review: Standardization of qPCR and RT-qPCR

New Guidelines Seek to Promote Accurate Interpretation of Data and Reliable Results

  • The perceived ease of use of real-time quantitative PCR (qPCR) and reverse transcription PCR (RT-qPCR) technology has revolutionized life science research. Its effectiveness at amplification and quantification of low levels of nucleic acids has driven the emergence of numerous applications, including cellular mRNA and miRNA quantification, biomarker discovery and validation, microbial quantification, cancer risk assessment, gene dosage determination, and detection of extremely low copy targets for forensic investigations.

    This, in turn, has resulted in an abundance of publications utilizing qPCR data obtained with diverse reagents, protocols, analysis methods, and reporting formats. Unfortunately, few papers report in detail how these results were obtained. This lack of clarity and transparency has led to concern in the research community over the reliability of qPCR data interpretation and the real danger of the scientific literature being corrupted with publications reporting erroneous and conflicting results.

    This has already occurred in some cases, resulting, for example, in retraction of a Science “Breakthrough of the Year 2005” report1. Now that qPCR has come of age, standardization is needed to ensure its validity, prompting the recent formulation of guidelines to increase experimental transparency, promote consistency between laboratories, and therefore, help assure the publication of valid conclusions2

  • Guidelines Proposed

    The Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines (MIQE) aim to provide a yardstick for the quality assessment of a publication. The MIQE guidelines define the minimum information required for evaluation of qPCR results, and they include a checklist to be included in the initial submission of a manuscript to a publisher.

    MIQE is modeled on similar guidelines such as MIAME (Minimal Information about a Microarray Experiment), developed several years ago3 and MIAPE (Minimal Information about a Proteomics Experiment)4. All of these are initiatives developed under the umbrella of the MIBBI (Minimum Information for Biological and Biomedical Investigations) standardization body, which has the goal of unifying all of the standardization guidelines for biological and biomedical research.

    In addition, the Real-Time PCR Data Markup Language (RDML) has been developed by a consortium to enable the straightforward exchange of qPCR data and related information between qPCR instruments and third-party data analysis software, between colleagues, and with journals or public data repositories5.

Readers' Comments

Posted 09/03/2009 by QPCR

Great that you disseminate the importance of this information. A lot of people are doing sloppy job on qPCR and their results are a problem by misleading people

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