SPECIAL REPORT -- Guidelines for Minimum Information for Publication of Quantitative Digital PCR Experiments.
Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, Hellemans J, Kubista M, Mueller RD, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT, Bustin SA.
Clin Chem 2013 59(6): 892-902
There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR). Consequently, dPCR has the potential to have a substantial impact on research as well as diagnostic applications. However, as with qPCR, the ability to perform robust meaningful experiments requires careful design and adequate controls. To assist independent evaluation of experimental data, comprehensive disclosure of all relevant experimental details is required. To facilitate this process we present the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines. This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation. Adoption of these guidelines by the scientific community will help to standardize experimental protocols, maximize efficient utilization of resources, and enhance the impact of this promising new technology.
Why the need for qPCR publication guidelines? - The case for MIQE
Stephen A. Bustin
Methods. 2010 April in qPCR special issue - The ongoing evolution of qPCR
Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry
Queen Mary University of London, Whitechapel, London E1 1BB, UK
The polymerase chain reaction (PCR) has matured from a labour- and time-intensive, low throughput qualitative gel-based technique to an easily automated, rapid, high throughput quantitative technology. Real-time quantitative PCR (qPCR) has become the benchmark technology for the detection and quantification of nucleic acids in a research, diagnostic, forensic and biotechnology setting. However, ill-assorted pre-assay conditions, poor assay design and inappropriate data analysis methodologies have resulted in the recurrent publication of data that are at best inconsistent and at worst irrelevant and even misleading. Furthermore, there is a lamentable lack of transparency of reporting, with the "Materials and Methods" sections of many publications, especially those with high impact factors, not fit for the purpose of evaluating the quality of any reported qPCR data. This poses a challenge to the integrity of the scientific literature, with serious consequences not just for basic research, but potentially calamitous implications for drug development and disease monitoring. These issues are being addressed by a set of guidelines that propose a minimum standard for the provision of information for qPCRexperiments ("MIQE"). MIQE aims to restructure to-day's free-for-all qPCR methods into a more consistent format that will encourage detailed auditing of experimental detail, data analysis and reporting principles. General implementation of these guidelines is an important requisite for the maturing of qPCR into a robust, accurate and reliable nucleic acid quantification technology.
Practical implementation of minimum standard guidelines for fluorescence-based
quantitative real-time PCR experiments
Stephen A Bustin, Jean-Francois Beaulieu, Jim Huggett, Rolf Jaggi, Frederick SB Kibenge, Pal A Olsvik,
Louis C Penning email and Stefan Toegel
BMC Molecular Biology 2010 - Published: 21 September 2010
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 precis) and guidance on the publication of pure 'reference gene' articles.
MIQE: Guidelines for the Design and Publication of a Reliable Real-time PCR Assay
Jim Huggett, Tania Nolan and Stephen A. Bustin
Real-Time PCR: Advanced Technologies and Applications
published 1st July 2013, Caister Academic Press
edited by Nick A. Saunders, Martin A. Lee
The capacity to amplify and detect trace amounts of nucleic acids has made the polymerase chain reaction (PCR) the most formidable molecular technology in use today. Its versatility and scope was further broadened first with the development of reverse transcription (RT)-PCR, which opened up the entire RNA field to thorough exploration and then, most conspicuously, with its evolution into real-time quantitative PCR (qPCR). Speed, simplicity, specificity, wide linear dynamic range, multiplexing and high-throughput potential, reduced contamination risk, simplified detection and data analysis procedures as well as availability of increasingly affordable instrumentation and reduced reagent cost have made qPCR the molecular method of choice when quantifying nucleic acids. Detection of pathogens, SNP analyses and quantification of RNA, even real-time analysis of gene expression in vivo have become routine applications and constant enhancements of chemistries, enzymes, mastermixes and instruments continue to extend the scope of qPCR technology by promising added benefits such as extremely short assay times measured in minutes, low reagent usage and exceptionally rapid heating/cooling rates. The whole process is driven by the insatiable demand for ever-more specific, sensitive, convenient and cost-effective protocols.
However, it has also become clear that variable pre-assay conditions, poor assay design and incorrect data analysis have resulted in the regular publication of data that are often inconsistent, inaccurate and often simply wrong. The problem is exacerbated by a lack of transparency of reporting, with the details of technical information wholly inadequate for the purpose of assessing the validity of reported qPCR data. This has serious consequences for basic research, reducing the potential for translating findings into valuable applications and potentially devastating implications for clinical practice. In response, guidelines proposing
a minimum standard for the provision of information for qPCR experiments (‘MIQE’) have been launched. These aim to establish a standard for accurate and reliable qPCR experimental design as well as recommendations to ensure comprehensive reporting of technical detail, indispensable conditions for the maturing of qPCR into a robust, accurate and reliable nucleic acid quantification technology.
Critical appraisal of quantitative PCR results in colorectal cancer research: Can we rely on published qPCR results?
J.R. Dijkstraemail address, L.C. van Kempen, I.D. Nagtegaal, S.A. Bustin
MOLECULAR ONCOLOGY (2014) 1-6
The use of real-time quantitative polymerase chain reaction (qPCR) in cancer research has become ubiquitous. The relative simplicity of qPCR experiments, which deliver fast and cost-effective results, means that each year an increasing number of papers utilizing this technique are being published. But how reliable are the published results? Since the validity of gene expression data is greatly dependent on appropriate normalisation to compensate for sample-to-sample and run-to-run variation, we have evaluated the adequacy of normalisation procedures in qPCR-based experiments. Consequently, we assessed all colorectal cancer publications that made use of qPCR from 2006 until August 2013 for the number of reference genes used and whether they had been validated. Using even these minimal evaluation criteria, the validity of only three percent (6/179) of the publications can be adequately assessed. We describe common errors, and conclude that the current state of reporting on qPCR in colorectal cancer research is disquieting. Extrapolated to the study of cancer in general, it is clear that the majority of studies using qPCR cannot be reliably assessed and that at best, the results of these studies may or may not be valid and at worst, pervasive incorrect normalisation is resulting in the wholesale publication of incorrect conclusions. This survey demonstrates that the existence of guidelines, such as MIQE, is necessary but not sufficient to address this problem and suggests that the scientific community should examine its responsibility and be aware of the implications of these findings for current and future research.
•Reliability-assessment of 179 qPCR studies in CRC-associated publications 2006–2013.
•Evaluation based on number of reference genes and their validation of suitability.
•97% of the qPCR-studies cannot be reliably assessed and results may not be valid.
•Guidelines, such as MIQE, are useful but by themselves are not sufficient.
•Scientific community should shoulder its responsibility.
The State of RT-Quantitative PCR: Firsthand Observations of Implementation of Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE)
Taylor SC, Mrkusich EM.
Bio-Rad Laboratories Canada, Mississauga, Ont., Canada.
J Mol Microbiol Biotechnol. 2013 Nov 28;24(1): 46-52
In the past decade, the techniques of quantitative PCR (qPCR) and reverse transcription (RT)-qPCR have become accessible to virtually all research labs, producing valuable data for peer-reviewed publications and supporting exciting research conclusions. However, the experimental design and validation processes applied to the associated projects are the result of historical biases adopted by individual labs that have evolved and changed since the inception of the techniques and associated technologies. This has resulted in wide variability in the quality, reproducibility and interpretability of published data as a direct result of how each lab has designed their RT-qPCR experiments. The 'minimum information for the publication of quantitative real-time PCR experiments' (MIQE) was published to provide the scientific community with a consistent workflow and key considerations to perform qPCR experiments. We use specific examples to highlight the serious negative ramifications for data quality when the MIQE guidelines are not applied and include a summary of good and poor practices for RT-qPCR.
Watch Taylor review the paper on YouTube
Standardisation and reporting for nucleic acid quantification
Jim Huggett & Stephen A. Bustin
Accred Qual Assur 2011
The real-time quantitative polymerase chain reaction (qPCR) is probably the most common molecular technique in use today, having become the method of choice for nucleic acid detection and quantification and underpinning applications ranging from basic research through biotechnology and forensic applications to clinical diagnostics. This key technology relies on fluorescence to detect and quantify nucleic acid amplification products, and its homogeneous assay format has transformed legacy polymerase chain reaction (PCR) from a low-throughput qualitative gel-based technique to a requently automated, rapid, high-throughput quantitative technology. However, the enormous range of protocols together with frequently inappropriate pre-assay conditions, poor assay design and unsuitable data analysis methodologies are impeding its status as a mature ,‘gold standard’ technology. This, combined with in consistent and in sufficient reporting procedures, has resulted in the wide spread publication of datat hat can be misleading, in particular when this tech-nology is used to quantify cellular mRNA or miRNA levels by RT-qPCR. This affects the integrity of the scientific literature, with consequences for not only basic research, but with potentially major implications for the potential development of molecular diagnostic and prognostic monitoring tools. These issues have been addressed by a set of guidelines that propose a minimum standard for the provision of information for qPCR experiments (‘MIQE’). MIQE aims to systematise current variable qPCR methods into a more consistent form at that will encourage detailed auditing of experimental detail, data analysis and reporting principles. General implementation of these guidelines is an important requisite for the maturing of qPCR into a robust, accurate and reliable nucleic acid quantification technology.
A MIQE-Compliant Real-Time PCR Assay for Aspergillus Detection
Johnson GL, Bibby DF, Wong S, Agrawal SG, Bustin SA.
Blizard Institute of Cell and Molecular Science, Queen Mary University, London, United Kingdom
PLoS One. 2012;7(7): e40022
The polymerase chain reaction (PCR) is widely used as a diagnostic tool in clinical laboratories and is particularly effective for detecting and identifying infectious agents for which routine culture and microscopy methods are inadequate. Invasive fungal disease (IFD) is a major cause of morbidity and mortality in immunosuppressed patients, and optimal diagnostic criteria are contentious. Although PCR-based methods have long been used for the diagnosis of invasive aspergillosis (IA), variable performance in clinical practice has limited their value. This shortcoming is a consequence of differing sample selection, collection and preparation protocols coupled with a lack of standardisation of the PCR itself. Furthermore, it has become clear that the performance of PCR-based assays in general is compromised by the inadequacy of experimental controls, insufficient optimisation of assay performance as well as lack of transparency in reporting experimental details. The recently published “Minimum Information for the publication of real-time Quantitative PCR Experiments” (MIQE) guidelines provide a blueprint for good PCR assay design and unambiguous reporting of experimental detail and results. We report the first real-time quantitative PCR (qPCR) assay targeting Aspergillus species that has been designed, optimised and validated in strict compliance with the MIQE guidelines. The hydrolysis probe-based assay, designed to target the 18S rRNA DNA sequence of Aspergillus species, has an efficiency of 100% (range 95–107%), a dynamic range of at least six orders of magnitude and limits of quantification and detection of 6 and 0.6 Aspergillus fumigatus genomes, respectively. It does not amplify Candida, Scedosporium, Fusarium or Rhizopus species and its clinical sensitivity is demonstrated in histological material from proven IA cases, as well as concordant PCR and galactomannan data in matched broncho-alveolar lavage and blood samples. The robustness, specificity and sensitivity of this assay make it an ideal molecular diagnostic tool for clinical use.
Editorial - Transparency of Reporting in Molecular Diagnostics
Stephen Bustin; Postgraduate Medical Institute, Anglia Ruskin University, Chelmsford CM1 1SQ, UK
Int. J. Mol. Sci. 2013, 14(8), 15878-15884
The major advances made over the past few years in molecular and cell biology are providing a progressively more detailed understanding of the molecular pathways that control normal processes and become dysregulated in disease. This has resulted in the documentation of numerous genetic, epigenetic, transcriptomic, proteomic and metabolomic biomarkers that promise earlier disease detection, more accurate patient stratification and better prognosis. Furthermore, molecular fingerprinting of diseases can be predictive of drug response and so assist with specific targeting of drugs against disease-associated molecules and function. ....
The MIQE Guidelines Uncloaked
Gregory L. Shipley
Chapter 8 in PCR Troubleshooting and Optimization: The Essential Guide, ISBN: 978-1-904455-72-1
Publication date - January 2011 Publisher: Caister Academic Press
Editors: Suzanne Kennedy and Nick Oswald MO BIO Laboratories, Inc., Carlsbad, CA 92010, USA and BitesizeBio, Edinburgh, UK
The MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines have been presented to serve as a practical guide for authors when publishing experimental data based on real-time qPCR. Each item is presented in tabular form as a checklist within the MIQE manuscript. However, this format has left little room for explanation of precisely what is expected from the items listed and no information on how one might go about assimilating the information requested. This chapter presents an expanded explanation of the guideline items with commentary on how those requirements might be met prior to publication.
Quantification noise in single cell experiments
Reiter M, Kirchner B, Müller H, Holzhauer C, Mann W, Pfaffl MW.
Nucleic Acids Res. 2011 Oct;39(18):e124
In quantitative single-cell studies, the critical part is the low amount of nucleic acids present and the resulting experimental variations. In addition biological data obtained from heterogeneous tissue are not reflecting the expression behaviour of every single-cell. These variations can be derived from natural biological variance or can be introduced externally. Both have negative effects on the quantification result. The aim of this study is to make quantitative single-cell studies more transparent and reliable in order to fulfil the MIQE guidelines at the single-cell level. The technical variability introduced by RT, pre-amplification, evaporation, biological material and qPCR itself was evaluated by using RNA or DNA standards. Secondly, the biological expression variances of GAPDH, TNFα, IL-1β, TLR4 were measured by mRNA profiling experiment in single lymphocytes. The used quantification setup was sensitive enough to detect single standard copies and transcripts out of one solitary cell. Most variability was introduced by RT, followed by evaporation, and pre-amplification. The qPCR analysis and the biological matrix introduced only minor variability. Both conducted studies impressively demonstrate the heterogeneity of expression patterns in individual cells and showed clearly today's limitation in quantitative single-cell expression analysis.
How to do successful gene expression analysis using real-time PCR
Derveaux S, Vandesompele J, Hellemans J.
Methods. 2010 50(4): 227-230 in qPCR special issue - The ongoing evolution of qPCR
Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.
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.
Improving the analysis of quantitative PCR data in veterinary researchComment on:
Bustin S, Penning LC.
Vet J. 2012 191(3): 279-281
Identification of internal control genes for quantitative expression analysis by real-time PCR in bovine peripheral lymphocytes. [Vet J. 2011]
Quantitative real-time PCR detection of insulin signalling-related genes in pancreatic islets isolated from healthy cats. [Vet J. 2010]
Common statistical and research design problems in manuscripts submitted to high-impact medical journals
Fernandes-Taylor S, Hyun JK, Reeder RN, Harris AH.
BMC Res Notes. 2011 Aug 19;4:304. doi: 10.1186/1756-0500-4-304.
Center for Health Care Evaluation, VA Palo Alto Health Care System and Stanford University School of Medicine, 795 Willow Road (MPD-152), Menlo Park, CA 94025, USA
BACKGROUND: To assist educators and researchers in improving the quality of medical research, we surveyed the editors and statistical reviewers of high-impact medical journals to ascertain the most frequent and critical statistical errors in submitted manuscripts.
FINDINGS: The Editors-in-Chief and statistical reviewers of the 38 medical journals with the highest impact factor in the 2007 Science Journal Citation Report and the 2007 Social Science Journal Citation Report were invited to complete an online survey about the statistical and design problems they most frequently found in manuscripts. Content analysis of the responses identified major issues. Editors and statistical reviewers (n = 25) from 20 journals responded. Respondents described problems that we classified into two, broad themes: A. statistical and sampling issues and B. inadequate reporting clarity or completeness. Problems included in the first theme were (1) inappropriate or incomplete analysis, including violations of model assumptions and analysis errors, (2) uninformed use of propensity scores, (3) failing to account for clustering in data analysis, (4) improperly addressing missing data, and (5) power/sample size concerns. Issues subsumed under the second theme were (1) Inadequate description of the methods and analysis and (2) Misstatement of results, including undue emphasis on p-values and incorrect inferences and interpretations.
CONCLUSIONS: The scientific quality of submitted manuscripts would increase if researchers addressed these common design, analytical, and reporting issues. Improving the application and presentation of quantitative methods in scholarly manuscripts is essential to advancing medical research.
Johnson G, Nolan T, Bustin SA.
Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK.
Methods Mol Biol. 2013 943: 1-16
Nucleic acids are the ultimate biomarker and real-time PCR (qPCR) is firmly established as the method of choice for nucleic acid detection. Together, they allow the accurate, sensitive and specific identification of pathogens, and the use of qPCR has become routine in diagnostic laboratories. The reliability of qPCR-based assays relies on a combination of optimal sample selection, assay design and validation as well as appropriate data analysis and the "Minimal Information for the Publication of real-time PCR" (MIQE) guidelines aim to improve both the reliability of assay design as well as the transparency of reporting, essential conditions if qPCR is to remain the benchmark technology for molecular diagnosis.
Improving biological relevancy of transcriptional biomarkers experiments by applying the MIQE guidelines to pre-clinical and clinical trials
Dooms M, Chango A, Barbour E, Pouillart P, Abdel Nour AM.
LaSalle Beauvais, 19 rue Pierre Waguet, 60 000 Beauvais, France.
Methods. 2013 59(1): 147-153
The "Minimum Information for the Publication of qPCR Experiments" (MIQE]) guidelines are very much targeted at basic research experiments and have to our knowledge not been applied to qPCR assays carried out in the context of clinical trials. This report details the use of the MIQE qPCR app for iPhone (App Store, Apple) to assess the MIQE compliance of one clinical and five pre-clinical trials. This resulted in the need to include 14 modifications that make the guidelines more relevant for the assessment of this special type of application. We also discuss the need for flexibility, since while some parameters increase experimental quality, they also require more reagents and more time, which is not always feasible in a clinical setting.
Cell-free microRNAs: potential biomarkers in need of standardized reporting
Michaela B. Kirschner, Nico van Zandwijk and Glen Reid
Asbestos Diseases Research Institute, University of Sydney, Sydney, NSW, Australia
Front. Genet., 19 April 2013
MicroRNAs are abundantly present and surprisingly stable in multiple biological fluids. These findings have been followed by numerous reverse transcription real-time quantitative PCR (RT-qPCR)-based reports revealing the clinical potential of using microRNA levels in body fluids as a biomarker of disease. Despite a rapidly increasing body of literature, the field has failed to adopt a set of standardized criteria for reporting the methodology used in the quantification of cell-free microRNAs. Not only do many studies based on RT-qPCR fail to address the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) criteria but frequently there is also a distinct lack of detail in descriptions of sample source and RNA isolation. As a direct result, it is often impossible to compare the results of different studies, which in turn, hinders progress in the field. To address this point, we propose a simple set of criteria to be used in conjunction with MIQE to reveal the true potential of cell-free microRNAs as biomarkers.
MIQE Guidelines in CHINESE
The MIQE Guidelines - Minimum Information for Publication of Quantitative Real-Time PCR Experiments
Stephen A. Bustin 1, Vladimir Benes 2, Jeremy A. Garson 3,4, Jan Hellemans 5, Jim Huggett 6, Mikael Kubista 7,8, Reinhold Mueller 9, Tania Nolan 10,
Michael W. Pfaffl 11, Gregory L. Shipley 12, Jo Vandesompele 5, and Carl T. Wittwer 13,14
Overseas Laboratory Medicine 2010: 3, 1
RDML: structured language and reporting guidelines for real-time quantitative PCR data.
Lefever S, Hellemans J, Pattyn F, Przybylski DR, Taylor C, Geurts R, Untergasser A, Vandesompele J; on behalf of the RDML consortium. Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.
Nucleic Acids Res. 2009 Apr;37(7): 2065-2069
Reliability of real-time reverse-transcription PCR in clinical diagnostics: gold standard or substandard?
Murphy J, Bustin SA.
Expert Rev Mol Diagn. 2009 9(2):187-197
Unreliable real-time PCR analysis of human endogenous retrovirus-W (HERV-W) RNA expression and DNA copy number in multiple sclerosis.
Garson JA, Huggett JF, Bustin SA, Pfaffl MW, Benes V, Vandesompele J, Shipley GL.
AIDS Res Hum Retroviruses. 2009 25(3): 377-378
Real-time polymerasechain reaction – towardsa more reliable, accurateand relevant assay.
EUROPEAN PHARMACEUTICAL REVIEW 2008 (6): 19-27
In-House Nucleic Acid Amplification Assays in Research: How Much Quality ControlIs Needed before One Can Rely upon the Results?
Petra Apfalter, UdoReischl and Margaret R. Hammerschlag
JOURNAL OF CLINICAL MICROBIOLOGY 2005 (dec): 5835–5841