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

ABSTRACT: 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.

SUBMITTER: Bustin SA

PROVIDER: S-EPMC2955025 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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

Bustin Stephen A SA Beaulieu Jean-François JF Huggett Jim J Jaggi Rolf R Kibenge Frederick S B FS Olsvik Pål A PA Penning Louis C LC Toegel Stefan S

BMC molecular biology 20100921

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 ...[more]

PMID: 20858237

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Project description:BackgroundCurrently real time PCR is the most precise method by which to measure gene expression. The method generates a large amount of raw numerical data and processing may notably influence final results. The data processing is based either on standard curves or on PCR efficiency assessment. At the moment, the PCR efficiency approach is preferred in relative PCR whilst the standard curve is often used for absolute PCR. However, there are no barriers to employ standard curves for relative PCR. This article provides an implementation of the standard curve method and discusses its advantages and limitations in relative real time PCR.ResultsWe designed a procedure for data processing in relative real time PCR. The procedure completely avoids PCR efficiency assessment, minimizes operator involvement and provides a statistical assessment of intra-assay variation. The procedure includes the following steps. (I) Noise is filtered from raw fluorescence readings by smoothing, baseline subtraction and amplitude normalization. (II) The optimal threshold is selected automatically from regression parameters of the standard curve. (III) Crossing points (CPs) are derived directly from coordinates of points where the threshold line crosses fluorescence plots obtained after the noise filtering. (IV) The means and their variances are calculated for CPs in PCR replicas. (V) The final results are derived from the CPs' means. The CPs' variances are traced to results by the law of error propagation. A detailed description and analysis of this data processing is provided. The limitations associated with the use of parametric statistical methods and amplitude normalization are specifically analyzed and found fit to the routine laboratory practice. Different options are discussed for aggregation of data obtained from multiple reference genes.ConclusionA standard curve based procedure for PCR data processing has been compiled and validated. It illustrates that standard curve design remains a reliable and simple alternative to the PCR-efficiency based calculations in relative real time PCR.

Dataset Information

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

Publications

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

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