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      MIQE qPCR and dPCR APP for iPhone, iPad and iPod - iOS Universal

New version available - Get help from a special team of experts in quantitative PCR (qPCR) and digital PCR (dPCR).
The MIQE APP helps you in reviewing scientific works and checking your own experiments, when qPCR is involved. Check your project's compliance to MIQE in minutes, have all required references to hand, and follow qPCR events and news ... ...
slide show

Over 7,500 downloads   => http://itunes.apple.com/app/miqe-qpcr/id423650002?mt=8






MIQE APP for
qPCR and dPCR
Update in July 2014


ANDROID MARKET MIQE qPCR and dPCR -- latest update from July 2014

Get help from a special team of experts in qPCR while on the move. MIQE - qPCR helps you in reviewing scientific works and checking your own experiments, when qPCR is involved. Check your project's compliancy to MIQE in minutes, have all required references in hands, and follow qPCR events and news.
  • The qPCR turns digital, so does this app. All new checklists, references dedicated to ddPCR are included!
  • Now MIQE qPCR app is even more interactive. For the first time ever, checklists are optimized in real time: you can reach 100% for every project if you are MIQE compliant.
  • Checklists are specific for each project type: just click on the kind of nucleic acid you are working with, the checklists adapts instantly by removing unnecessary items (Reverse transcription items are not relevant if working only on DNA for instance).
  • Moreover, some items may not apply to your specific experiments. You can now remove them and have the most accurate MIQE compliancy.
  • References have been updated, so you can keep in touch with latest MIQE related literature, symposium updates and more.
  • Last but not least: Export is available. You can archive the state of your project whenever you want, share it with your colleagues, and store it.



Since May 2012 a html5 version of the MIQE_qPCR APP is online
=> MIQE-html5.gene-quantification.info

To run this application you need a html5 compatible web browser!


MIQE Guidelines - Chinese translation MIQE Guidelines - Japanese translation MIQE Guidelines - Arabian translation

2014
  • microRNA Quality Control Study:
    Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study

    Nature Methods 11, 809–815 (2014)
    MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.
  • Real-Time PCR: Yet More Worlds to Conquer
    by Mary Ann Labant
    The polymerase chain reaction (PCR), invented about three decades ago, soon entered mainstream use thanks to an ongoing series of refinements.
    One particularly important refinement, introduced about two decades ago, is the “real time” quantification of DNA. The idea is to trace the rising level of DNA throughout the amplification step, and not just measure the final amount of amplified product. This idea turns standard PCR into real-time PCR, or quantitative PCR.
    Real-time PCR has become the most widely used nucleic acid detection technology. It is routinely used in academic research, in applied testing settings such as food-safety or veterinary testing, and in molecular diagnostics. It continues to replace many older detection methods due to simple readouts, high sensitivity, and multiplex and quantification capabilities, as well as ease of use, cost effectiveness, and throughput flexibility with only moderate equipment investments................
  • Quantitative PCR (qPCR) and the guide to good practices MIQE: adapting and relevance in the clinical biology context
    Dooms M, Chango A, Abdel-Nour A [Article in French]
    Ann Biol Clin (Paris). 2014 72(3): 265-269
    The qPCR has been introduced in clinical and biomedical research for over 10 years from now. Its use in trials and diagnostics is continuously increasing. Due to this heavy use, the question of relyability and relevance of qPCR results has to be asked. This review proposes a documented and evidence based answer to this question, thanks to the MIQE (minimum information for publication of quantitative real-time PCR experiments) guideline. The whole analysis process is addressed, from nucleic acids extraction to data management. Simple answers are given, taking into account the technical constraints from clinical research in order to allow a realistic application of this guideline.
  • Why are reporting guidelines so essential?
    by LGC in July 2014
    We’ve all probably heard the Chinese proverb that it’s not the destination that is important but the journey. Well the same can be said of scientific research: it’s not only the results that matter but the methodology and processes that lead us to them.Scientist carries out analysis on genetically modified organism using polymerase chain reaction (PCR)OK, so this may be a little facetious but the concept is not and it’s the reason why guidelines for the reporting of scientific experiments have been emerging over the past decade across scientific disciplines – including the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.PCR (polymerase chain reaction) is a technique widely used in molecular biology to identify and quantify DNA. PCR works by targeted amplification of DNA by several orders of magnitude to enable identification and measurement of specific sequences. Quantitative polymerase chain reaction (qPCR), also called real-time polymerase chain reaction, is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule.In order to encourage increased transparency in reported data, the MIQE guidelines were developed and published in 2009. But why is this so important and, five years later, what impact have the guidelines had?

  • ANDROID MARKET MIQE qPCR and dPCR -- latest update from July 2014
    Get help from a special team of experts in qPCR while on the move. MIQE - qPCR helps you in reviewing scientific works and checking your own experiments, when qPCR is involved. Check your project's compliancy to MIQE in minutes, have all required references in hands, and follow qPCR events and news.
    • The qPCR turns digital, so does this app. All new checklists, references dedicated to ddPCR are included!
    • Now MIQE qPCR app is even more interactive. For the first time ever, checklists are optimized in real time: you can reach 100% for every project if you are MIQE compliant.
    • Checklists are specific for each project type: just click on the kind of nucleic acid you are working with, the checklists adapts instantly by removing unnecessary items (Reverse transcription items are not relevant if working only on DNA for instance).
    • Moreover, some items may not apply to your specific experiments. You can now remove them and have the most accurate MIQE compliancy.
    • References have been updated, so you can keep in touch with latest MIQE related literature, symposium updates and more.
    • Last but not least: Export is available. You can archive the state of your project whenever you want, share it with your colleagues, and store it.
  • New MIQE 2014 papers by the MIQE research community:
    Minimum Information Necessary for Quantitative Real- Time PCR Experiments
    Gemma Johnson, Afi f Abdel Nour, Tania Nolan, Jim Huggett, and Stephen Bustin
    Roberto Biassoni and Alessandro Raso (eds.), Quantitative Real-Time PCR: Methods and Protocols, Methods in Molecular Biology, vol. 1160
    Springer Science+Business Media New York 2014

    Five Years MIQE Guidelines: The Case of the Arabian Countries
    Afif M. Abdel Nour, Esam Azhar, Ghazi Damanhouri, Stephen A. Bustin
    PLoS ONE 9(2) (2014): e88266
    Toward Enhanced MIQE Compliance: Reference Residual Normalization of qPCR Gene Expression Data
    Richard C. Edmunds, Jenifer K. McIntyre, J. Adam Luckenbach, David H. Baldwin, and John P. Incardona
    JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 25, ISSUE 2, 0 2014
  • “Stay in touch while on the bench” - how the MIQE applet can increase the quality of your qPCR and dPCR experiments
    Afif M Abdel Nour, Esam Azhar, Michael W Pfaffl and Ghazi Damanhouri
    http://www.biomedcentral.com/1471-2164/15/S2/P15
    Background - How to check the quality of your qPCR experiments or the reliability of an international publication by an electronic device? From now on iOS based mobile devices, e.g. iPhone, iPad or iPod, will help you to increase the quality of qPCR experiment or publication, by providing a ‘MIQE (The MIQE Guidelines - Minimum Information for publication of Quantitative real-time PCR Experiments) qPCR’ applet [1,2]. Three years ago we created the first interactive solution for scientific guidelines, based on the MIQE qPCR publication [3].
    Materials and methods - This first applet was downloaded 7800 times from 89 different countries, and recently Biotechniques journal has selected the APP as one of the leading “methods-oriented applets” and recommended it by the “websites every life scientist should try”.
    Results - After this great success we are presenting today the MIQE applet for the digital PCR (Figure 1).
    The digital PCR MIQE guidelines were published recently by a group of experts [4]. This new application could be used by scientists to check whether their digital PCR (dPCR) experiment or the used literature fulfills the MIQE requirement. The ‘MIQE Guidelines’ checklist provides 83 parameters that dPCR studies should be required or recommended to meet before being considered for publication. This checklist is based on the original published MIQE checklist for the dPCR and we hope it will increase future publication quality and reliability.
    Conclusions - There are much more wider seen goals of the ‘MIQE guidelines’, all in all the goals might be summarized as follow: 1) to increase reliability of results, 2) to help insuring the integrity of scientific work, with major focus on biological relevance. This is an easy to use applet that can help reviewers and authors to gain time in there manuscript preparation. Recently by using this applet we managed to analyze and evaluate 461 scientific papers published by Arabian countries (paper accepted in PLOSOne).
    Figure 1. Screenshots from the iOS applet.


  • Design and Validation of Real-Time PCR Primers
    Bio-Rad collaborated with Biogazelle, leaders in real-time PCR research, to design and experimentally validate PCR primers for gene expression assays across the human and mouse transcriptomes. All PCR primers were designed to meet stringent performance standards following the MIQE guidelines (minimum information for publication of quantitative real-time PCR experiments; Bustin et al. 2009).
  • qPCR and MIQE Seminar Series
    Sigma Aldrich Learning Center
    As part of our customer education program, we have provided two recorded seminar series covering the topics of qPCR and MIQE. The recorded sessions are intended to provide a high level overview of these subject matters. We have kept the lessons concise so that you can enjoy a self-paced learning program.
  • The MIQE Bedtime Story: A Tale of Two qPCR Experiments
    Posted: January 31, 2014, by Sean Taylor, Joey St-Pierre and Cathy Vaillancourt
    Kelly and Ted were two budding Ph.D. students from different labs in fierce competition and required the use of qPCR (quantitative PCR) to support results generated from microarray and systems biology data. They were both working on two genes implicated in neural regeneration and a compound postulated to induce proliferation of neurons. It was clear from the posters they presented at a recent conference that they were working on the same project and that the associated data would be well received by the scientific and medical community with a publication in a high-profile journal. This also meant that one of the students was going to get scooped, which only served to highlight the need to produce the qPCR data quickly, and unfortunately neither student had experience with this technique.

  • MIQE Guidelines for RT qPCR Experiments
    13 Jan 2014
    Scientists and journals have been slow to adopt the Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines that were established in 2009 to bolster the reliability of real-time PCR (qPCR) and reverse transcription qPCR (RT-qPCR) data. To help boost adoption, Bio-Rad scientists published a brief and practical guide that concisely summarizes the key steps required to produce high-quality, reproducible data for labs conducting RT-qPCR experiments.
    We spoke co-author of the guide, Bio-Rad Field Application Specialist, Sean Taylor, to understand more the importance of MIQE, the low adoption rates and what the future may hold. http://www.youtube.com/watch?v=_LI-lH-mgvI

2013
  • The State of RT-qPCR -- Firsthand Observations of MIQE Implementation
    Follow on YouTube
  • MIQE-Richtlinien bei quantitativer Real Time PCR (German)
    Die Bedeutung der MIQE-Guidelines bei der quantitativen Real Time PCR (qPCR) zur Detektion und Quantifizierung von Nukleinsäuren unterstreicht ein aktuelles Paper in "Nature Methods". Die MIQE-Richtlinien wurden 2009 von einer Gruppe internationaler WissenschafterInnen aufgesetzt, um die Qualität, Richtigkeit, Überprüfbarkeit und Zuverlässigkeit gewonnener qPCR-Ergebnisse im Labor und in der wissenschaftlichen Literatur zu steigern. Tögel: "Die quantitative Real Time PCR zählt zu den am häufigsten verwendeten molekularbiologischen Methoden." Die qPCR wird in der Grundlagenforschung ebenso eingesetzt wie in der Entwicklung von Pharmazeutika oder bei der Erkennung von Erbkrankheiten oder Virusinfektionen.
  • A Brief RT-qPCR “Field Guide” for MIQE Adherence
    09 Dec 2013 by Bio-Rad
    Scientists and journals have been slow to adopt the Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines that were established in 2009 to bolster the reliability of real-time PCR (qPCR) and reverse transcription qPCR (RT-qPCR) data. To help boost adoption, Bio-Rad scientists Sean Taylor and Eli Mrkusich, have published a brief and practical guide that concisely summarizes the key steps required to produce high-quality, reproducible data for labs conducting RT-qPCR experiments.

  • Surveys Indicate Studies Still Eschew Good qPCR Practices Despite Evidence that MIQE Helps
    November 07, 2013 by Ben Butkus
    A pair of surveys examining more than 1,700 peer-reviewed publications whose authors used quantitative real-time PCR has revealed a lack of transparent and comprehensive reporting of essential technical information — and that the most prestigious scientific journals are the biggest offenders.
    The surveys also showed that the number of papers citing MIQE — a set of qPCR experiment guidelines devised by concerned researchers — is still far outnumbered by those not citing the guidelines; however, those that do cite MIQE show the improved transparency and reporting of technical details necessary for high-quality, reproducible results.
    As qPCR has grown ubiquitous as a research and molecular diagnostic tool for detecting and quantifying nucleic acids and gene expression, many researchers have been guilty of taking the technique for granted. Multiple publications have demonstrated that qPCR results are only meaningful if a number of complex technical steps and quality control provisions are taken; however, there have been many instances of published, peer-reviewed papers that shirked these necessary steps, resulting in ambiguous or downright meaningless qPCR data.



The need for transparency and good practices in the qPCR literature

Stephen A Bustin, Vladimir Benes,Jeremy Garson, Jan Hellemans, Jim Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W Pfaffl, Gregory Shipley, Carl T Wittwer, et al.
Nature Methods 2013, 10(11): 1063–1067
Published online 30 October 2013


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.

The MIQE guidelines aim “to encourage better experimental practice and more transparent reporting, resulting in more reliable, comparable and unequivocal interpretation of qPCR results”. They are a response to the considerable misgivings with which many researchers perceive the quality of published qPCR data. That unease comes as a surprise to those who incorrectly believe that the conceptual simplicity and accessibility of qPCR translates into an equally uncomplicated experimental procedure.

In reality, it is very easy to publish qPCR results that are meaningless. Without transparency for optimization, validation and quality-control procedures, it is impossible for the reader of a publication to distinguish a reliable from a biased result or technical variation. This is particularly true for protocols aimed at quantifying RNA targets using reverse transcription qPCR (RT-qPCR), for which the relevance of the results is critically dependent on sampling procedure, sample properties, template quality and analysis procedures in addition to any relevant qPCR parameters.

  

  • Getting over qPCR's technical hurdles
    Sarah Webb, Ph.D. BioTechniques, Vol. 55, No. 4, October 2013, pp. 165–168
    Even though it has become a “workhorse” technique for quantifying nucleic acids, qPCR continues to be plagued by problems of reproducibility and reliability. Yet when carefully designed, optimized, and validated, qPCR experiments are incredibly accurate, according to Stephen Bustin of Anglia Ruskin University in Chelmsford, UK. However, in far too many cases, researchers don't carefully optimize and validate their assays or report enough information on reagents, primers and procedures for the research community to evaluate their methods.
    When Bustin started using qPCR for gene expression profiling in cancer metastases during the late 1990s, he quickly discovered the pitfalls and problems associated with qPCR data— going so far as to write an early review article bringing the issues out in the open. As a result, he was asked to serve as an expert witness in a high profile UK court case where his analysis of qPCR data showed flaws that wrongly linked the MMR vaccine with autism.
    Years of discussions focusing on these technical challenges amongst Bustin and like-minded colleagues eventually culminated in a 2009 article published in the journal Clinical Chemistry that provides a set of guidelines for performing qPCR experiments. These guidelines are known as the minimum information for publication of quantitative real-time PCR experiments (MIQE) (1). Four years later though, only 11%of papers that report qPCR experiments cite those guidelines.

  • MIQE_qPCR applet is the winner
    Ten Apps for Busy Researchers: PCR
    MIQE_qPCR is the winner
    We continue our series exploring digital science tools by presenting apps focused on PCR

  • Ten Methods-oriented Apps and Websites Every Life Scientist Should Try
    09/19/2013
    Our exploration of the world of digital science tools continues this week with a collection of methods-oriented sites and apps that should prove useful to all biomedical researchers. From worms to molecular cloning and qPCR to cataloguing plasmid sequences on your iPhone, these tools show how digital tech in the lab can be both fun and useful. Learn more...

  • New MIQE APP available
    Bio-Rad Sponsors New Version of MIQE qPCR App for Real-Time PCR and Digital PCR
    miqe appBio-Rad Laboratories is sponsoring a new version of the MIQE qPCR app. Researchers can use the new version to validate their digital PCR (dPCR) experiments according to the recently published digital MIQE (dMIQE) guidelines
    (Huggett et al. 2013).
    Professors Michael Pfaffl (TU Munich, Weihenstephan, Germany) and Afif Nour (King Abdulaziz University, Saudi Arabia) designed the original MIQE qPCR app to help researchers improve their real-time PCR (qPCR) assay protocols by making it easier to adopt a set of best practices described in an earlier publication: The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments
    (Bustin et al. 2009). Progress bars in the app show the percentage of assay compliance as each item in the MIQE checklist is completed.
    “We were pleased to find that the MIQE qPCR app encouraged our customers to follow the new MIQE guidelines,” said Jean-Pierre Dakkak, a laboratory equipment trading company manager.
    “Researchers are really eager to learn how this app can make their life easier,” said Dr. Nour. “They report it instills confidence in the validity of every qPCR or dPCR project.”
    The new updates include:
    - Project-specific checklists — checklists remove unnecessary items; for example, reverse transcription items are irrelevant in DNA research
    - Updated references — researchers can stay current with the latest MIQE and dMIQE literature and qPCR symposiums
    - Easy export — users can save their projects and share them with colleagues
    The MIQE qPCR app runs on the Apple iPhone, iPod touch, and iPad. It has been downloaded more than 6,500 times in 87 countries. To get your copy, visit http://bit.ly/MIQEapp

FREE Webinar -Wednesday September 11, 2013 - 2:30 - 4:00 p.m. Eastern Time
Register Now

Thirty years ago, in 1983, Kary B. Mullis conceived an experimental method for amplifying small quantities of DNA— the polymerase chain reaction (PCR)—that would go on to revolutionize the study of genetics, forensics, and biological anthropology. Over three decades, PCR techniques, fueled by advances in enzymology and automation, have continually improved and evolved to meet the changing needs and demands of life-science researchers. Today, armed with an arsenal of potent reagents, reliable software, and robust instrumentation, PCR will be a vital part of new applications of next-generation sequencing, clinical diagnostics, and drug discovery.

The Scientist, in collaboration with Biosearch Technologies, has invited Kary Mullis to reflect back on these 30 years in terms of his initial discovery, how things stand today, and where he thinks PCR is headed in the future. For this live webinar, Dr. Mullis will be joined by expert panelists who will highlight current innovations taking place in real-time qPCR in terms of the technology and its applications. They will also discuss the impact of next-generation PCR technologies such as digital PCR, direct PCR, immuno PCR and more.


  • 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.   ....
  • A comparison of miRNA isolation and RT-qPCR technologies and their effects on quantification accuracy and repeatability.
    Redshaw N, Wilkes T, Whale A, Cowen S, Huggett J, Foy CA.
    LGC Limited, Queens Road, Teddington, Middlesex, UK.
    Biotechniques. 2013 54(3): 155-164
    MicroRNAs (miRNAs) are short (~22 nucleotides), non-coding RNA molecules that post-transcriptionally regulate gene expression. As the miRNA field is still in its relative infancy, there is currently a lack of consensus regarding optimal methodologies for miRNA quantification, data analysis and data standardization. To investigate miRNA measurement we selected a panel of both synthetic miRNA spikes and endogenous miRNAs to evaluate assay performance, copy number estimation, and relative quantification. We compared two different miRNA quantification methodologies and also assessed the impact of short RNA enrichment on the miRNA measurement. We found that both short RNA enrichment and quantification strategy used had a significant impact on miRNA measurement. Our findings illustrate that miRNA quantification can be influenced by the choice of methodology and this must be considered when interpreting miRNA analyses. Furthermore, we show that synthetic miRNA spikes can be used as effective experimental controls for the short RNA enrichment procedure.
  • Follow the MIQE presentations
    You’ll find here all the MIQE and QC records held at qPCR & NGS Events in the past three years – qPCR 2010 in Vienna, qPCR 2011 and qPCR & NGS 2013 in Freising-Weihenstephan => www.econferences.de/miqe-talks/

Nucleic Acids Research Group (NARG)
| Current Members | Studies | Electronic Posters | Protocols | Publications | Links | Membership History |

The Nucleic Acids Research Group is focused on studying current topics related to any and all aspects of nucleic acids including protocols, sample preparation, and storage. Most recently the focus has been on nucleic acid degradation and detection of epigenetic-related nucleic acids. Membership is open to all interested ABRF members.

Biorepositories:  Considerations for sample acquisition, storage, and QC

Recent studies conducted by the Nucleic Acid Research Group (NARG) have focused on the impact of sample degradation on biological assays. While this is important, we have yet to address the major factors related to storage and proper handling in a Biorepository setting. During the 2012 ABRF conference, a roundtable session to discuss all aspects of storage and biobanking will be guided by panel of experts in the areas of DNA, RNA and protein biobanking. Data from a detailed survey conducted among ABRF members using biorepositories will be presented with information related to sample acquisition, sample storage, and the types of quality control (QC) measures will be presented.

  • My MIQE Guide - Empowering results that matter
    published in June 2013 by Roche Applied Science
    Improved reproducibility. Faster publication. More trustworthy data. Get the most out of your gene expression research with the help of the MIQE guidelines for qPCR.
    What are the MIQE guidelines?
    This website is designed to help you along every step of your workflow – from isolation to reverse transcription to analysis – while making it easier to collect the information necessary to make your research publication-ready and fully MIQE-compliant.
  • Cell-free microRNAs: potential biomarkers in need of standardized reporting
    Front. Genet., 19 April 2013  by  Michaela B. Kirschner, Nico van Zandwijk and Glen Reid
    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 for the Design and Publication of a Reliable Real-time PCR Assay
    from Jim Huggett, Tania Nolan and Stephen A. Bustin writing in Real-Time PCR: Advanced Technologies and Applications:
    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.

  • MIQE and qPCR quality concerns
    2nd May 2013 by TATAA Biocenter
  • MIQE qPCR Guidelines -- Three Years Later
    05/01/2013 in Biotechniques by Lauren Arcuri Ware
    Three years ago, a group of researchers began campaigning for the adoption of guidelines that promised to improve the reproducibility of RT-qPCR data. Lauren Arcuri Ware reports on the adoption and evolution of these guidelines.
  • Do TaqMan® Assays Comply with MIQE Guidelines? -- Ask TaqMan
    Published on Feb 25, 2013
    Submit your real-time PCR questions at  http://www.lifetechnologies.com/asktaqman
    After introducing MIQE guidelines, a set of standards for producing and publishing qPCR data, Sr. Field Applications Specialist Doug Rains explains how Life Technologies' large and diverse collection of TaqMan® assays comply with these guidelines. Learn about Life Technologies' TaqMan assays guarantee program and the rigorous bioinformatics process through which each TaqMan® assays is designed.
  • qPCR and MIQE Seminar Series
    Sigma Aldrich Learning Center
    As part of our customer education program, we have provided two recorded seminar series covering the topics of qPCR and MIQE. The recorded sessions are intended to provide a high level overview of these subject matters. We have kept the lessons concise so that you can enjoy a self-paced learning program.
  • Nature Methods - Technology Feature Top - PCR:  living life amplified and standardized
    by Vivien Marx in Nature Methods May 2013 (10) 5: pp391 - 395
    With strategies for reproducibility and quality control, scientists seek to cultivate better practices in quantitative PCR experiments.



  • 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 Apr 9. [Epub ahead of print]

    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.
  • CONGRATULATIONS to all MIQE authors
    1st April 2013
    Today the MIQE paper reached more than 1400 citations!
    http://scholar.google.de/scholar?cites=6338124712618390161&as_sdt=2005&sciodt=0%2C5&hl=de
    http://www.clinchem.org/content/55/4/611.short

  • Going the MIQE way  --  Reporting Checklist For Life Sciences Articles  (download PDF)
    This checklist is used to ensure good reporting standards and to improve the reproducibility of published results. For more information, please read Reporting Life Sciences Research (PDF).
  • CHALLENGES IN IRREPRODUCIBLE RESEARCH
    No research paper can ever be considered to be the final word, and the replication and corroboration of research results is key to the scientific process. In studying complex entities, especially animals and human beings, the complexity of the system and of the techniques can all too easily lead to results that seem robust in the lab, and valid to editors and referees of journals, but which do not stand the test of further studies. Nature has published a series of articles about the worrying extent to which research results have been found wanting in this respect. The editors of Nature and the Nature life sciences research journals have also taken substantive steps to put our own houses in order, in improving the transparency and robustness of what we publish. Journals, research laboratories and institutions and funders all have an interest in tackling issues of irreproducibility. We hope that the articles contained in this collection will help.

  • Announcement:   Reducing our irreproducibility
    NATURE | EDITORIAL -- Nature 496, 398 (25 April 2013)
    Over the past year, Nature has published a string of articles that highlight failures in the reliability and reproducibility of published research. The problems arise in laboratories, but journals such as this one compound them when they fail to exert sufficient scrutiny over the results that they publish, and when they do not publish enough information for other researchers to assess results properly.
    From next month, Nature and the Nature research journals will introduce editorial measures to address the problem by improving the consistency and quality of reporting in life-sciences articles. To ease the interpretation and improve the reliability of published results we will more systematically ensure that key methodological details are reported, and we will give more space to methods sections. We will examine statistics more closely and encourage authors to be transparent, for example by including their raw data.
    Central to this initiative is a checklist intended to prompt authors to disclose technical and statistical information in their submissions, and to encourage referees to consider aspects important for research reproducibility. It was developed after discussions with researchers on the problems that lead to irreproducibility, including workshops organized last year by US National Institutes of Health (NIH) institutes. It also draws on published concerns about reporting standards (or the lack of them) and the collective experience of editors at Nature journals.
    The checklist is not exhaustive. It focuses on a few experimental and analytical design elements that are crucial for the interpretation of research results but are often reported incompletely. For example, authors will need to describe methodological parameters that can introduce bias or influence robustness, and provide precise characterization of key reagents that may be subject to biological variability, such as cell lines and antibodies. The checklist also consolidates existing policies about data deposition and presentation.
    We will also demand more precise descriptions of statistics, and we will commission statisticians as consultants on certain papers, at the editor’s discretion and at the referees’ suggestion.
    We recognize that there is no single way to conduct an experimental study. Exploratory investigations cannot be done with the same level of statistical rigour as hypothesis-testing studies. Few academic laboratories have the means to perform the level of validation required, for example, to translate a finding from the laboratory to the clinic. However, that should not stand in the way of a full report of how a study was designed, conducted and analysed that will allow reviewers and readers to adequately interpret and build on the results.
    To allow authors to describe their experimental design and methods in as much detail as necessary, the participating journals, including Nature, will abolish space restrictions on the methods section.
    To further increase transparency, we will encourage authors to provide tables of the data behind graphs and figures. This builds on our established data-deposition policy for specific experiments and large data sets. The source data will be made available directly from the figure legend, for easy access. We continue to encourage authors to share detailed methods and reagent descriptions by depositing protocols in Protocol Exchange (www.nature.com/protocolexchange), an open resource linked from the primary paper.
    Renewed attention to reporting and transparency is a small step. Much bigger underlying issues contribute to the problem, and are beyond the reach of journals alone. Too few biologists receive adequate training in statistics and other quantitative aspects of their subject. Mentoring of young scientists on matters of rigour and transparency is inconsistent at best. In academia, the ever increasing pressures to publish and chase funds provide little incentive to pursue studies and publish results that contradict or confirm previous papers. Those who document the validity or irreproducibility of a published piece of work seldom get a welcome from journals and funders, even as money and effort are wasted on false assumptions.
    Tackling these issues is a long-term endeavour that will require the commitment of funders, institutions, researchers and publishers. It is encouraging that NIH institutes have led community discussions on this topic and are considering their own recommendations. We urge others to take note of these and of our initiatives, and do whatever they can to improve research reproducibility.

  • 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
  • Real-time quantitative PCR, pathogen detection and MIQE
    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
  • PrimePCR and MIQE
    January 23, 2013 at Canadian Biotechnologist
    The MIQE guidelines have set a new standard for publishing qPCR results to ensure integrity in the scientific literature and increase experimental transparency. One of the key guidelines focuses on the proper design and experimental validation of primer assays used in qPCR. This tech report describes the design and wet-lab validation of PrimePCR assays and how they comply with the MIQE guidelines.
    Read   PrimePCR™ Assays: Meeting the MIQE Guidelines by Full Wet-Lab Validation
  • MIQE Trouble-Free
    Real-Time PCR
    can be tough. It requires careful planning and much optimization. When it works, you feel great. When it fails…fill in the blank. There are times in our research career when we feel like giving up. Nothing we do seems to yield positive results. Then…along comes a kit. Sure, at first we are wary about using a kit. After all, weren’t we put on this planet to troubleshoot and suffer through tortuous experiments? Alas, many of us quickly overcome that guilt and put our trust and faith in the hands of others. But how do we know that commercially available kits are indeed trustworthy? Perhaps they too will yield erroneous results and lead us down the dark path of non-publishable gobbledygook data. So what do we do? We troubleshoot. We troubleshoot the commercially available kit. The kit that we purchased to avoid troubleshooting! Curses! It’s one thing to troubleshoot my own experimental protocol, but to troubleshoot someone else’s? And one that I paid for nonetheless?
    Well, fellow scientists, feel the pain no more. At least not in the world of qPCR. Bio-Rad Laboratories has teamed up with leaders in Real-Time PCR to bring you the most robust, commercial qPCR kit on the market, PrimePCR. Bio-Rad’s PrimePCR™ Assays and Panels have been designed to meet MIQE criteria (we shouldn’t have to tell you what that is, just see our previous posts “A practical approach to MIQE for the bench scientist” and “Applications of MIQE to real time quantitative PCR” among other posts) and incorporate the following key requirements:
    • high assay specificity without the use of a probe
    • compatibility with standard assay conditions
    • avoided secondary structures in primer annealing sites
    • avoided SNPs in target regions
    • maximized fraction of transcript isoforms being detected
    • designed intron-spanning assays whenever possible
    • used latest release of genome builds and annotation databases

Moreover, the kits have undergone wet-lab validation at the hands of PCR professionals so you don’t have to waste precious time validating and troubleshooting an assay that you spent money on acquiring.
To learn more about Bio-Rad’s Prime PCR Assays read PrimePCR™ Assays: Meeting the MIQE guidelines by full wet-lab validation

2012
  • Scientific Integrity: Can Molecular Medicine Be Trusted
    September 2012
    The aim of presentaton is to allow the audience who are not likely to be expert in the PCR field, to come away with a clear understanding why there is absolutely no chance of any of the scientific data being correct.
    A 1998 Lancet publication reported on the case histories of 12 previously normal children who developed symptoms of autism or inflammatory bowel disease after having had received the measles, mumps, and rubella (MMR) vaccine. This paper formed the basis for claims of a new type of gastrointestinal disease, termed autistic enterocolitis and sparked a major health scare in the UK and the USA.The lecture will review the complete absence of credibility associated with the molecular evidence underlying a supposed link between the MMR vaccination and autism in children.     Download the slides

  • MIQE Guideline List In French
    by Primerdesign
    Le guide MIQE:  Informations minimales pour la publication d’articles liés à la RT-PCR Quantitative
  • PrimePCR™ Assays: Meeting the MIQE Guidelines by Full Wet-Lab Validation
    Pubished 20th August 2012
    Quantitative PCR (qPCR) is a powerful tool in life science research however the data it generates can only be trusted if sufficient attention is paid to quality control and assurance at every step of the experimental workflow.  Bio-Rad’s PrimePCR assays are a unique and attractive alternative to computer-based assay design methods.  Every PrimePCR assay has been wet-lab validated to ensure data integrity using rigorous and state-of-the-art laboratory testing procedures including verification with next generation sequencing (NGS).  Read the tech report.
  • Genetic Engineering & Biotechnology News - Expert Tips
    10 Tips to Improve Your qPCR and RT-qPCR Results
    Get the best results from your real-time PCR assays with these tips.
    by Terri Sundquist , Gabriela Saldanha

  • CONGRATULATIONS to all MIQE authors
    1st October 2012
    Today the MIQE paper reached more than 1000 citations!
    http://scholar.google.de/scholar?cites=6338124712618390161&as_sdt=2005&sciodt=0%2C5&hl=de
    http://www.clinchem.org/content/55/4/611.short

  • New MIQE qPCR eSeminar - A sensitive and specific qPCR assay is a basic prerequisite for the acquisition of reliable, reproducible and biologically relevant results
    Register for our live eSeminar: Wednesday, October 10, 2012  4:30 pm (Central Europe)
    Speaker: Prof. Stephen Bustin, Allied Health and Medicine at Anglia Ruskin University
    Learn how easy it is to design assays!
    Many published assays have been neither appropriately designed nor properly validated, leading to conclusions that are frequently wrong.
    Some commercial vendors offer predesigned assays, claiming to generate optimal results for negligible user input. The quality of these assays varies considerably and it remains essential to optimize and validate those assays in laboratories.
    Consequently, the ability to design assays remains a key skill for anyone aiming to use qPCR assays and the aim of this seminar is to demonstrate just how simple good assay design is.
    Join this live eSeminar and learn about:
    •  How to streamline a workflow that allows researchers to design bespoke assays
    •  Develop techniques to generate robust data.

  • MIQE Guidelines:  A Roadmap for Proper qPCR Experimental Design and Reporting
    Presented by Professor Stephen Bustin
    Tuesday, September 25th. 2:00 pm
    Tuesday, September 25th. 8:00 pm
    * Times are in your local timezone.
    Quantitative real-time PCR (qPCR) is today’s most widely used molecular technology, and continuous improvements to chemistries, enzymes, master mixes, and instruments have led to an increasingly reliable assay that virtually guarantees results. However, the same improvements that make it easy to generate qPCR data often conceal experimental practices that have resulted in the regular publication of data that are inconsistent, inaccurate and, sometimes, simply wrong. Incomplete reporting of experimental detail further confounds assessment of qPCR data validity, calling into question scientific conclusions that serve as a basis for further basic research and diagnostic applications.
    Learn how to:
    - Design, evaluate, and report your qPCR experimental data according to the MIQE Guidelines.
    - Enable the reproducibility of your experiment.

  • Bio-Rad Launches MIQE-Compliant qPCR Assays and Panels Validated with Biogazelle
    August 30, 2012 by Ben Butkus
    Bio-Rad this week introduced qPCR assays and panels that have been wet-lab validated to meet the minimum information for publication of quantitative real-time PCR experiments, or MIQE, guidelines.
  • MIQE Guidelines Qualitätskontrolle in der real-time RT-qPCR (in German)
    3-4 May 2012 - 2nd Life Science Conference Analytik Jena, Germany (from spectronetvideo)
    Prof. Dr. Michael W. Pfaffl - Technische Universität München
    • Central problem areas in qPCR
    • Pre-PCR RNA processing
    • Validation of total RNA integrity
    • Degradation gradient
    • Influence on RIN value on mRNA expression
    • Quantification Strategies in real time qRT-PCR
    • intra-assay & inter-assay variation
    • Determination of RT real-time PCR efficiency
    • The use of reference genes
  • A MIQE-Compliant Real-Time PCR Assay for Aspergillus Detection
    July 2012 - PLoS ONE 7(7): e40022
    Gemma L. Johnson, David F. Bibby, Stephenie Wong, Samir G. Agrawal, Stephen A. Bustin 
    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.
  • How to Produce Conclusive, Reproducible RT-qPCR Data Every Time
    Jun 17, 2012;  Cathy Vaillancourt, Ph.D., and Sean Taylor, Ph.D.
    Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is everywhere. Its ubiquity, however, does not mean its practitioners produce consistent and reproducible results that reflect the underlying biology. The exquisite sensitivity of RT-qPCR demands rigorous experimental design; the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were published to help standardize this technique [1, 2]. Although a growing number of labs are applying the MIQE criteria to their experiments, many groups still ignore them, thus increasing the potential of publishing artifactual and inaccurate data. In fact, the erroneous conclusions from previous studies have not only misdirected research but have also had poignant societal impact [for example, see ref. 3, 4].
    RT-qPCR employs four principal steps:
    1. Sample extraction from cells or tissue in a rigorous and reproducible manner;
    2. RNA isolation and purification;
    3. Reverse transcription to convert the mRNA to cDNA;
    4. The qPCR reaction to quantify levels of reverse transcribed cDNA in the sample.
  • New Study Demonstrates False Conclusions Reached if MIQE Not Followed in Human Placenta Quantitative PCR Studies
    05 Jul 2012
    Inappropriate selection of reference genes and degraded RNA can lead to flawed data and erroneous results in reverse transcription quantitative PCR (RT-qPCR) studies. Scientists at INRS-Institut Armand Frappier in Laval, Quebec, in collaboration with Bio-Rad Laboratories, Inc., came to these conclusions in a paper published recently in Molecular Biotechnology.

  • Real-Time PCR Experiment Guidance
    Jun 15 | Tools And Technology
    The first MIQE qPCR app, sponsored by Bio-Rad Laboratories Inc., provides researchers with resources and checklists needed to ensure MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) compliance for qPCR experiments.
The Future of qPCR - Best practices, Standardization, and the MIQE Guidelines
link to video

This event occurred on Thursday, September 30, 2010
download transcript
download extended transcript
download slides

  • Definitive qPCR Book Series
    June 2012 edited by Stephen Bustin
    These iBooks are available for download on your iPad with iBooks 2 or on your computer with iTunes. Download links:
    Vol 1: Assay Design - http://itunes.apple.com/gb/book/definitive-qpcr/id509231219?mt=11
    Vol 2: Basic Principles - http://itunes.apple.com/gb/book/definitive-qpcr/id535566436?mt=11
    Vol 3: Nucleic Acids QC - coming in August 2012
  • Fast Accurate PCR and qPCR
    Want fast, accurate PCR and qPCR results? Look no further than Rainin FrameStar™ PCR plates. FrameStar™ technology combines thin-walled polypropylene wells with rigid, thermally-stable polycarbonate plate frames for superior performance.
  • Drivers and Hurdles for qPCR
    by Mikael Kubista
    Genetic Engineering News Feature Articles: May 1, 2012 (Vol. 32, No. 9)
    According to a recent report on the gene-amplification technologies market from Global Industry Analysts, there are close to 70,000 bioresearchers using real-time quantitative PCR (qPCR) in North America alone. They spend $740 million annually on instruments and reagents. Annual growth, notes the market report, is about 16% and may even increase as the major hurdle for small companies trying to enter the field—basic PCR technology patent protection—expired last year. The global market is forecast to reach $1.9 billion by 2015.

  • How Reliable is Real-Time PCR?
    2. May 2012 by Sarah C.P. Williams in Biotechniques
    Two people can perform the same real-time PCR experiment and get different results. Researchers are quantifying this variability to understand its source and how to fix it.
  • MIQE is entering the Chinese market!
    编辑推荐
    两个人完成同样的实时PCR(real-time-PCR)实验有可能得到不同的结果。研究人员正在量化这一差异 性以了解它的来源以及如何解决这一问题。
  • A Conversation About qPCR with Jo Vandesompele
    Dr Jo Vandesompele is a professor of functional genomics and applied bioinformatics at Ghent University, where his lab primarily focuses on cancer genomics. He is also an internationally recognized expert on quantitative PCR and co-founder, together with Dr Jan Hellemans, of the biotechnology company Biogazelle. Biogazelle offers products and services to assist researchers with all aspects of performing real-time PCR, from experimental design and data generation through comprehensive, user-friendly data analysis. We recently had the opportunity to speak with Dr Vandesompele about his research, Biogazelle, and the field of qPCR.
    What is the significance of the MIQE guidelines?
    I am quite happy that there is so much attention given to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines [3]. The initiative to produce the MIQE guidelines was started by Dr Stephen Bustin, and was the work of an unofficial consortium of qPCR experts who were all frustrated with the problems with qPCR methods in published papers. For example, it is very common that there is not enough information to repeat the experiment. In addition, investigators do not always address everything that needs to be accounted for in a proper qPCR experiment, or you cannot tell if they did because the details are not sufficiently reported. These issues are critical, as professor Bustin rightly points out, because they actually corrupt the integrity of the literature with data that is of questionable quality.
    The importance of these concerns in published qPCR studies compelled us to summarize the most essential criteria that should be addressed and reported when setting up a qPCR experiment. The resulting 85 parameter checklist should help researchers document and perform better qPCR experiments. Investigators can find information on the guidelines and partners at the MIQE website.
    We do understand that the guidelines were developed by academic groups doing research, and that they may not be appropriate for all fields and applications such as clinical diagnostics, digital PCR, and genotyping. This is why the consortium needs input from the community, so we can design extended or modified checklists for specific applications or fields.
    How do you think MIQE will affect qPCR reagent suppliers?
    MIQE is all about transparency and the ability to replicate studies. However, there have been extensive discussions in the consortium about what the minimum requirements for transparency should be. Based on the original MIQE paper, one might argue that authors who are using products from companies that do not provide primer and probe sequences are not complying with MIQE standards, and the consortium might want to prevent such companies from selling their products or promote some vendors over others. In an ideal world it is probably correct that we should report all of the relevant experimental information. However, some vendors have said that they cannot provide all of this information, and we have to recognize that we live in a commercial environment where we have to find compromise between intellectual property and the ability to replicate an experiment.
    This is why we came up with a consensus paper that states that while it is still recommended to report primer sequences, it is not absolutely required [4]. Instead, the newly modified standard says that providing a context sequence that can be used to identify the applicable amplicon sequence +/-15 bases is sufficient, as long as it allows others to replicate the experiment.
    It is interesting to note that many commercial suppliers of qPCR tools are introducing their products as MIQE compliant. It demonstrates that these companies see the importance of what we are trying to accomplish with the guidelines and want to promote them. With that in mind, it is important to not give too much weight to suppliers of tools who state their product is MIQE compliant. It probably means that the product is a useful tool in the qPCR workflow, but it does not really add an extensive quality label to that product.
    With that in mind, I do appreciate that some qPCR suppliers, like IDT, do provide all the recommended information, including primer and probe sequences. It is preferable that companies do that and they should be rewarded somehow for providing detailed information, at  least through appreciation from the scientific community.

  • Sean and Frank: Kings of the MIQE
    April 2012
    Since time immemorial, (or at least stretching back 2 to 3 years…), the American Biotechnologist has been a staunch advocate of the MIQE standards for real-time qPCR and has presented videos, technotes and papers from Bio-Rad qPCR experts. Dr. Sean Taylor’s video “Applications of MIQE to Real Time Quantitative PCR” has become and instant internet sensation and Dr. Francisco Bizouarn’s slideshow on “Fast qPCR assay optimization and validation techniques for HTS” is enshrined in the SlideShare museum hall of fame (at least on our site). Now, these two world class scientists have finally gotten the recognition they deserve.
    This week, Sean and Frank were interviewed by the PCR Insider regarding the importance of following MIQE when conducting qPCR studies and Bio-Rad’s role in the dissemination of these crucial guidelines.

Welcome to Stephen Bustin's publishing website
an iTunes eBook series  Definitive qPCR  edited by Stephen Bustin

An exhaustive guide to assay design for quantitative real-time PCR. The book describes the basic concepts important for amplicon selection and primer and probe design. There are step-by-step examples for designing probe-based and SYBR Green assays targeting mRNA and fungal pathogens using several popular design programs. These are then exposed to extensive in silico analysis to identify the optimum amplicon/primer/probe combination. There is a detailed trouble shooting guide, a listing of instruments, reagents and additional information available on the internet, all with hyperlinks. In addition, there are three Keynote presentations summarising the main concepts of standard assay design, multiplex assay design and explaining the rationale behind MIQE, the guidelines for qPCR publication transparency.

more eBook
  • Einladung zur 2. Life Science Conference 2012
    Analytik Jena lädt Sie herzlich zur »2. Life Science Conference« vom 03. bis 04. Mai 2012 nach Jena ein.
    Erleben Sie eine Reihe hochinteressanter wissenschaftlicher Vorträge zur Molekulardiagnostik und zu aktuellen Forschungsthemen. Namhafte Referenten und Experten der Branche geben Ihnen Einblicke in aktuelle Trends und wissenschaftliche Themen rund um das Produktportfolio von Analytik Jena | Life Science.
    Unsere Top-Themen im Überblick
       MIQE Guidelines – Qualitätskontrollee in der Real-Time RT-qPCR
       Prof. Dr. Michael W. Pfaffl, Technische Universität München
       EHEC Diagnostik O104:H4 - Der Keim im Fokus
       Dr. Ulrich Busch, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL)
       Ultraschneller DNA-Nachweis mit Nanopartikeln
       Dr. Lars Ullerich, GNA Biosolutions GmbH, München
  • Takara Bio Europe Offers Real-Time PCR Training Webinars in Collaboration with World-Class Provider, TATAA Biocenter
    February 2012
    Takara Bio Europe today announced that it has joined forces with leading qPCR training provider TATAA Biocenter to offer free educational webinars in real-time PCR (qPCR) for academic and industrial researchers and laboratory technicians.
    Takara Bio Europe President Jean-Jacques Farhi explained the reasons for the offering: “We believe that consistent and comparable PCR data can only be generated by a combination of well-designed experiments and high-quality reagents, such as those in the Takara/Clontech range. For this reason, we have collaborated with TATAA Biocenter, the world’s premier molecular technique training organiser, to offer training webinars that will allow attendees to address key questions in designing qPCR experiments.”
    TATAA Biocenter has over 20 years’ experience in qPCR training and was directly involved in drawing up the ‘Minimum Information for Publication of Quantitative Real-Time PCR Experiments’ (MIQE) guidelines designed to improve data reliability.
    Professor Mikael Kubista, CEO and founder of TATAA Biocenter explained that the courses will be delivered online through audiovisual webinars. They will be aimed at helping beginners to overcome the major hurdles of the qPCR technique. He said, “To begin with, the complexity of most biological samples makes designing experiments and protocols challenging. We will be available throughout the webinar to address questions and give feedback to participants on specific cases.”
    Participants in the webinars will also be tested on their newly-acquired knowledge with prizes of either a year’s supply of Takara Bio qPCR reagents (up to 5000 rxns), or a place in a qPCR wet lab course at TATAA Biocenter, with contributions to travel costs for the “top of the class”.

  • Q&A: Bio-Rad Scientists Discuss Case Study Demonstrating MIQE Importance in qPCR Experiments
    February 29, 2012 by Bio-Rad
    First published in 2009 in the journal Clinical Chemistry, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines — better known as the MIQE guidelines — were designed to provide researchers with a roadmap for improving the quality and reliability of their qPCR data
    Since that time, the molecular biology research community has slowly adopted the guidelines, and many qPCR instrument and reagent vendors have done their part to help encourage their customers to follow MIQE protocols.
    There is still work to be done, however, and some vendors have taken a more active role than others in disseminating information about MIQE to their customers. To wit, Bio-Rad earlier this month published a case study on its website demonstrating how neglecting some of the key steps in the MIQE guidelines can lead to flawed data and erroneous conclusions.
    In the study, researchers from Bio-Rad and the Jewish General Hospital at McGill University studied the effect of RNA sample quality and reference gene stability on gene expression data obtained using qPCR.
    More specifically, they used the minichromosome maintenance protein MCM7 as a model target gene to investigate the importance of appropriate reference gene selection. They also varied RNA sample quality from their breast cancer samples to determine its effect on data.
    Following the MIQE guidelines, they observed a significant increase in gene expression of MCM7 between normal and tumor samples when using high-quality and high-purity RNA with normalization using stable reference genes. However, they obtained inconclusive and even opposite results when using poor-quality RNA samples and unstable reference genes.
  • MIQE qPCR  --  Best Of The Web:   Feb 2012 (Vol. 32, No. 3)
    Rated  * * * => VERY GOOD => Nice user interface, strong reference resource
    Disadvantages => Nothing major
    Quantitative Polymerase Chain Reaction—more affectionately known as qPCR—has become a staple in many molecular biology labs. For researchers who use qPCR, an equally important acronym is MIQE, which stands for the Minimum Information for Publication of Quantitative Real-Time PCR Experiments. MIQE guidelines are in place to ensure the accuracy, interpretability, and reproducibility of published qPCR experiments. So, are your experiments in compliance? You can easily monitor your experiments using the MIQE qPCR app. This app allows one to create projects and update checklists corresponding to MIQE guidelines. Project information can also be exported. In addition, the app contains references to current literature regarding MIQE, experimental design, sample preparation, nucleic acid extraction, protocols, and other pertinent qPCR topics. Thus, this app both allows researchers to monitor their experiments and to keep up-to-date on the latest qPCR news.
  • A practical approach to RT-qPCR - Publishing data that conform to the MIQE guidelines
    Methods Vol 50, Issue 4, Pages S1-S5
    by Sean Taylor, Michael Wakem, Greg Dijkman, Marwan Alsarraj, Marie Nguyen
  • Aiming to Optimize qPCR Steps
    by Michael D. O'Neill
    Progress in real-time quantitative PCR (qPCR) technology has been steady since its invention approximately 15 years ago. Recent innovations and where the technology is headed in the future will be discussed at a Select Biosciences’ upcoming conference on “Advances in qPCR”.
  • Special: PCR -   Qualitätsmanagement in der RT-qPCR
    Für die quantitative Real Time PCR (RT-qPCR) zeigen Catrin Wernicke, Philipp Franke, Lars Radke, Stephan Berge und Marcus Frohme die Kriterien, die Probleme und die möglichen Lösungswege für eine standardisierte Etablierung von Genexpressionsanalysen in den Life Sciences auf.
    Several aspects in the numerous steps of a reverse transcription (RT) quantitative PCR may interfere with the result’s validity. Therefore, before starting the proper investigation, a particular assay establishment is required.

  • Design and Optimization of qPCR Experiments According to the MIQE Guidelines to Assure Reproducible and Quantifiable Results
    A McGill Channels Event;  Event Date:  Thu, 2012-02-16 at 16:00
    by Dr. Sean Taylor, Bio-Rad Laboratories, will present the second lecture in the Winter series of 4 O'Clock Forum.
    4 O'Clock Forum is a monthly seminar series held on the Macdonald campus where researchers and graduate students have regular opportunities to be exposed to scientific advancements related to their own fields of research as well as other scientific areas. Light refreshments will be served.  ALL VISITORS WELCOME!
  • MIQE and RDML Guidelines
    by Bio-Rad
    Overview - Real-time quantitative PCR (qPCR) has become a definitive technique for quantification of differences in gene expression levels between samples. Over the past 10 years, the popularity of this method has grown exponentially, with the publication of well over 25,000 papers from diverse fields of science. Apart from the broad applicability of the technique, one of the central factors that have stimulated its impressive growth is the increased demand from journal review panels for the use of RT-qPCR to support phenotypic observations with quantitative, molecular data. Furthermore, gene expression analysis is now being used to support protein expression data from proteomics-based assays.
    In this section we discuss MIQE guidelines that define the minimum information that needs to be provided when publishing qPCR experiments. We also describe RDML an XML-based markup language created for the consistent reporting of real-time PCR experiments.
    Page Contents:
    - Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines
    - Real-Time PCR Data Markup Language (RDML)
    - Practical MIQE Tools for Researchers
    - References
  • Sigma Aldrich TV
    The MIQE Assay Design Considerations by Tania Nolan
  • When Do Guidelines Become Requirements?  -  MIQE Makes a Difference in PCR
    By George Rodrigues, Ph.D., Senior Scientific Manager, Artel
    A guideline such as Minimum Information for Publication of Quantitative Real-time PCR Experiments (MIQE) is a good example of how a science-based guideline can impact customers and suppliers, becoming a kind of “voluntary regulation” which aims to improve the quality of laboratory research. Compliance with this guideline allows researchers and laboratories to regulate themselves, rather than wait for government to impose a standard on them which may be unnecessarily restrictive and/or inappropriate. As well, use of the guideline can have marketing benefits to the company using it – by positioning themselves as quality-minded.
    Analytical methods built around real time or quantitative polymerase chain reaction (PCR) technology are widespread and of increasing importance in many fields.  Supporting the development, acceptance and transparency of this technology is an ongoing effort to provide a guideline titled “Minimum Information for Publication of Quantitative
    Real-time PCR Experiments” also known as MIQE.
    MIQE (pronounced mykee) includes a checklist for both essential and desirable information which should be included in research papers pertaining to PCR.  This information allows reviewers and readers to evaluate the quality of the research work and also aids other researchers when they attempt to repeat the results. 
    So at what point does a guideline become requirement?  In the case of MIQE guidelines, they have been adopted by the American Association for Clinical Chemistry and have become an expectation for authors submitting research papers to the association’s scientific journal Clinical Chemistry.  Others such as Oxford Journals have also adopted this guideline in its policy for authors.
    On the business side, the makers of PCR reagents and systems have found a marketing advantage in claiming that their products are “MIQE complaint”.
    The checklist itself contains 57 essential items and 28 desirable items. One of these desirable items (but not essential) is the disclosure of probe sequences. Not all vendors disclose this information, so disclosure was not made a requirement. However, a footnote to the list makes it clear that use of products where sequence is not disclosed “is discouraged”. This shows how even a non-mandatory recommendation has the potential to adversely impact product acceptance.
    A guideline such as MIQE is a good example of how a science-based guideline can impact customers and suppliers and become a kind of “voluntary regulation” which aims to improve the quality of laboratory research.  Compliance with this guideline allows researchers and laboratories to regulate themselves, rather than wait for government to impose a standard on them.
    To this author it seems that voluntary adoption of science-based quality guidelines are an attractive way for laboratory professionals to take the initiative to improve quality and avoid the imposition of mandatory regulations which may be less attractive than simply volunteering to do the right thing.
  • A Conversation About qPCR with Jo Vandesompele
    qPCR dataanalysis - qBASEplus - MIQE guidelines

    Dr Jo Vandesompele is a professor of functional genomics and applied bioinformatics at Ghent University, where his lab primarily focuses on cancer genomics. He is also an internationally recognized expert on quantitative PCR and co-founder, together with Dr Jan Hellemans, of the biotechnology company Biogazelle. Biogazelle offers products and services to assist researchers with all aspects of performing real-time PCR, from experimental design and data generation through comprehensive, user-friendly data analysis. We recently had the opportunity to speak with Dr Vandesompele about his research, Biogazelle, and the field of qPCR.
  • The MIQE guidelines are part of the Bitnos - 'Biomedical Guidelines'
    1. Exp Anim Guidelines
    2. MIQE: Minim. Informat. Real-time RT-PCR
    3. GLP Experimental animals
    4. GLP in Pdf format/FDA
    5. GLP (OECD)
    6. Recombinant DNA Guidelines (NIH)
    7. MicroArray Quality Control (MAQC)
  • Minimizing Variation in qPCR Workflows - Two Novel Tools Help Reduce Variability and Improve Accuracy
    Ian Kavanagh
    Tutorials: Jan 1, 2012 (Vol. 32, No. 1)
    Large numbers of drug discovery projects involve the analysis of gene-expression levels to accurately assess target effects. When screening compounds from a library, it is common to use microarray techniques since they provide a broad overview of genome-wide expression levels. Microarrays do have their limitations, however—while they can generate a broad portfolio of data on a single chip, there are sometimes discrepancies over the precision of the expression levels of each individual gene.
    Before progressing along the drug discovery pipeline, it is key that microarray results are effectively validated. Real-time quantitative polymerase chain reaction (qPCR) is often used as the final step in any microarray protocol to ensure the accuracy and repeatability of the data prior to further analysis.
    The drug discovery process in its entirety can be both time-consuming and costly. In order to streamline this into an efficient workflow, accuracy at every step is essential. The most promising initial hits need to be identified and the least promising candidates eliminated early on. Therefore, microarray data needs to be reliable, and validation via qPCR is a logical quality-control step.
    However, qPCR itself has its own challenges, with well-to-well and plate-to-plate variability impacting the accuracy of the quantification of expression. Users need to be confident that reaction uniformity is maintained across each plate throughout an entire PCR run.
    In this article, we discuss the use of two Thermo Fisher Scientific products - the Thermo Scientific RNA Spike Control and PikoReal Real Time PCR thermal cycler - as part of a molecular biology workflow to reduce variability when amplifying target sequences.

2011



MIQE Guidelines slowly entering "high impact" journals!





LIFE SCIENCE TECHNOLOGIES - qPCR Innovations and Blueprints
7. October 2011  by Chris Tachibana  in  ScienceMag.org   PDF version
Quantitative PCR users can rapidly generate large amounts of high-quality data with new instruments and products made possible by microfluidics and miniaturization technology. These platforms are the tools for developing techniques that require extremely high throughput and sensitivity such as digital PCR and single-cell analysis. Researchers are adopting these methods to ask sophisticated questions about genetics and cancer biology as well as to develop novel research and diagnostic assays. As qPCR innovators explore new frontiers and everyday users venture into more complicated workflows, international groups of industry and academic partners are keeping us on the path of best practices. Two consortia (MIQE & SPIDIA) are generating guidelines on the qPCR process - from experimental design and pre-analysis sample collection, to processing data and publishing results. The guidelines are blueprints that ensure reproducibility, validity, and transparency.





LIFE SCIENCE TECHNOLOGIES - Gene-Expression Analysis Exploits More Technologies
Science - November 2011  PDF version
To quantify the expression of specific genes, researchers can use a variety of techniques, including arrays, PCR, and high throughput sequencing. However, getting accurate results still depends on precisely carrying out these methods, even with increasingly user-friendly technologies. In fact, as more scientists study gene expression, the standards for analysis are growing more rigorous to ensure that only accurate data are published. Likewise, software has been keeping pace, helping researchers follow protocols and analyze their results.





qPCR  -  quicker and easier but don't be sloppy
by Monya Baker  Nature Methods  8, 207–212 (2011)   PDF version
Gene profiling using quantitative PCR is becoming higher throughput, but researchers must be careful in gathering their data.
Stephen Bustin knew something was wrong as soon as he visited the laboratory. He was investigating reports that using a technique called real-time quantitative PCR (qPCR) researchers had identified measles virus in intestinal tissue of children with developmental disorders1. If true, those results supported the theory that a commonly administered vaccine caused autism in young children. If not, anxieties of parents and public health officials had been needlessly inflamed. Bustin found that this laboratory was next door to a facility producing DNA plasmids - a likely source of contamination. Even worse, on at least two occasions the researchers had neglected a basic step. Because measles virus is made of RNA, it must be converted to DNA before PCR can work. The enzyme that effects this conversion, reverse transcriptase, had been left out of some protocols, but there was no change in the results. Whatever they had detected was certainly not measles virus.




Routine lab method's accuracy called into question
Nature Medicine Vol 16, page 349 (2010)  by  Catherine Shaffer  in 
Nature Medicine  
PDF version
In 2002, four years after first sparking public controversy over whether the measles, mumps and rubella vaccine causes autism, Andrew Wakefield reported a possible molecular mechanism for the connection. He claimed that a form of irritable bowel disease, which he called autistic enterocolitis, was triggered by the measles virus (Molec. Pathol. 55, 84–90, 2002). That finding, however, was based on a “defective experimental technique,” Stephen Bustin, a molecular biologist at Barts and the London School of Medicine and Dentistry, told a US federal court in 2007. The problem: Wakefield had incorrectly applied the common laboratory protocol known as quantitative real-time polymerase chain reaction (qPCR) to come to his conclusions.
Bustin says this faulty lab work is a problem shared by many researchers around the world who have turned to qPCR to measure gene expression. Unlike standard PCR, which can only crudely quantify levels of DNA, the chemistry behind qPCR allows researchers to assess such levels more precisely by comparing sequences of interest against a known reference added to the test tube mix as a control.
But the reference genes used in qPCR can vary between experiments and laboratories, which can give misleading results or make it difficult to compare one study to another. As a result of this and other variables in the technique, a majority of scientific papers involving qPCR include flawed methods, say a team of leading qPCR experts. Most qPCR methods, as reported in the literature, are improperly validated and irreproducible, Bustin claims.
Last year, he and 11 colleagues published a set of more than 60 individual standards - collectively called the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to address this problem ( Clin. Chem. 55, 611–622, 2009 ). “If you look at the literature, you find again and again and again the appalling quality of qPCR protocols,” says Bustin, who this month repeated his call for the scientific community to adopt the MIQE guidelines (Methods 50, 217–226, 2010). “There's no excuse for anyone either not reporting or not doing experiments properly.”
The consequence of poor methodology is that many published papers contain erroneous conclusions, says Mikael Kubista, a coauthor of the MIQE guidelines and chief executive of the TATAA Biocenter in Göteborg, Sweden. “The problem is that the technique itself seems so simple and so easy to do, (but) in real life you're analyzing biological samples with complexity.”


  • A MIQE Case Study — Effect of RNA Sample Quality and Reference Gene Stability on Gene Expression Data
    Published: December 15, 2011; by Sean Taylor, Bio-Rad Laboratories Canada, 1329 Meyerside Dr Mississauga, ON, L5T 1C9, and Marguerite Buchanan and Mark Basik, McGill University, Jewish General Hospital, Montreal, QC
    Published: December 15, 2011
    Abstract - Real-time quantitative PCR (qPCR) has become the gold standard for validating DNA microarray data and is routinely used to determine gene expression differences between a wide variety of samples. The exquisite sensitivity of the technology permits the detection of a single copy of a target gene in a sample which has led to qPCR now being used in the clinical setting to diagnose infection and disease states. In an effort to standardize the design of the associated experiments, the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines were published in 2009. In this study, we show how qPCR can lead to erroneous conclusions regarding differences in MCM7 gene expression between normal and tumor human breast cancer samples if the key steps set out in the MIQE guidelines are not followed.

  • RESULTS: Delivering solutions across an entire workflow solution
    What is RESULTS? RESULTS is a program designed to deliver solutions across an entire workflow solution -- from sample collection to data collection. Additionally, it provides unparalleled access to the brands and technical innovation you need today.
    The analysis performed in your lab every day rely on one thing ... a result. Whether you are working on complex research, reporting QC data for product release or isolation of a gene of interest, you rely on results to make decisions, execute your next move and take you to the next step of a process or project. At Fisher Scientific, we recognise the importance of data quality and are committed to helping you achieve accurate, reliable results every time.
    The evolution of scientific technology makes it essential for you to have access to brands, technical expertise and the latest products to ensure your success. Our goal is to deliver the right product solution to solve your research challenges and improve productivity -- allowing you to focus on what's most important, the science.
    To achieve our goal, Fisher Scientific developed RESULTS, a program designed to deliver solutions across an entire workflow solution -- from sample collection to data collection. Additionally, it provides unparalleled access to the brands and technical innovation you need today.
    RESULTS applications include Proteomics, Genomics, Cell Biology, Microbiology and Separation Science.

  • Un enseignant-chercheur co-développe une application iPhone pour la qPCR.
    La qPCR, vous connaissez ? Il s’agit d’une méthode d’analyse des acides nucléiques (ADN et ARN), notamment appliquée dans le domaine alimentaire pour quantifier des ADN (bactéries, OGM…) présents dans un aliment et pouvant en affecter sa qualité, et en médecine, pour détecter des cellules cancéreuses, des mutations génétiques ou encore des malformations de fœtus.
    Depuis 2009, un référentiel incontournable destiné aux chercheurs, le MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments), liste les informations minimales à insérer dans une publication exposant des expériences réalisées par qPCR. A la demande de la société Bio-Rad (leader dans le domaine de la biologie moléculaire), Afif Abdel Nour, enseignant-chercheur en biologie moléculaire, et Michael Pfaffl, de la Technical University of Munich, en ont développé sa version numérique, interactive, sous forme d’une application pour iPhone et iPad intitulée MIQE_qPCR.
    Le principe est simple : l’application liste, par thématique, l’ensemble des items du référentiel MIQE à aborder dans la publication. Le chercheur coche les items à mesure qu’il avance dans ses écrits, lui permettant de voir l’avancement de son projet en temps réel. Une bibliographie sur MIQE ainsi que la possibilité de partager le projet en cours sont des services complémentaires inclus dans l’application.

  • In search of better real-timePCR data
    by Richard Kurtz, In Drug Discovery & Development - 1st October 2011
    Real-time quantitative PCR (qPCR) has become the industry standard for the detection and quantification of nucleic acids. However, the lack of consensus among researchers on how to best perform and interpret qPCR experiments is a major hurdle for advancing the technology. This problem is exacerbated when insufficient experimental detail is given in published work, impeding the ability of others to accurately evaluate or replicate reported results
    .
  • The StellARray® system supports adherence to MIQE guidelines
    Autumn 2011 by Martina Reiter in Lonza Resource Notes page 12-13
    The increasing number of citations of the MIQE guidelines (minimum information for publication of quantitative real-time PCR experiments, Bustin et al., 2009) demonstrates a growing emphasis on standardized experimental practice for qPCR. Lonza’s SYBR-based StellARray® qPCR array system offers a simple and reliable system for gene expression analysis that meets the MIQE standards and makes it easier for qPCR users to compare and publish their results.
  • MIQE Guidelines for publishing - Solaris qPCR Gene Expression Assays
    Thermo Fisher Scientific  Copyright © 2011
    Solaris qPCR Gene Expression Assays are MIQE compliant, in particular because sequence information for the assay is provided.
    The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were published in 2009 and aim to encourage better experimental practice so that published qPCR data accurately reflects the true biological picture. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments.
    The MIQE guidelines focus on the reliability of results to help ensure the accuracy of scientific literature, promote consistency between laboratories, and increase experimental transparency. The paper includes a checklist to support the submission of a manuscript to publishers/journals. By providing all relevant experimental conditions and assay characteristics, reviewers are better placed to assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results.
    MIQE details should be published either in abbreviated form or as an online supplement.
    Checking that inhibition is not affecting RT-qPCR data is one way to follow the MIQE guidelines. Checking for inhibition reduces the likelihood of reporting inaccurate or incorrect data and conforms to MIQE guidelines, and the Solaris RNA Spike Control Kit is a simple and effective way to achieve this.
  • Posts tagged ‘MIQE’
    October 2011 by Canadian BioTechnologist 2.0
    Are you using the right reference genes?
    Considering Real-Time PCR for gene expression analysis? Have you tested multiple reference genes or are you just going to run with your favorite such as 18S? Check out this article on suitable reference gene selection before you move forward. It could save you lots of grief in the long run.
    Applications of MIQE to Real Time Quantitative PCR
    In this video, Dr. Sean Taylor, Field Applications Specialist, Bio-Rad Laboratories, demonstrates how sample quality and reference gene selection effect data analysis and interpretation in real-time quantitative PCR (qPCR) experiments. The presentation is in accordance with the previously published MIQE guidelines.
    For enhanced viewing, click on the full-screen mode button on the bottom right hand corner of the video.
    A Practical Approach to MIQE for the Bench Scientist
    In a groundbreaking review published in February 2009, Bustin et al bemoaned the lack of standardization in Quantitative Real-Time PCR (qPCR) experimentation and data analysis. In their critique the authors cite the use of diverse reagents, protocols, analysis methods and reporting formats which has negatively impacted on the acceptance of qPCR as a robust quantitative molecular tool. The most serious technical deficiencies include:
        * sample storage
        * sample preparation
        * sample quality
        * choice of primers and probes
        * inappropriate data and statistical analysis
  • Step up to the MIQE
    Drug Discovery - Issue 18 - by Richard Kurtz
    “Quality data is paramount for the successful development and potential approval of a drug candidate”
    When it comes to real-time PCR in drug discovery, Richard Kurtz believes that MIQE guidelines will help create a clear path to better results.
    Polymerase chain reaction (PCR) has evolved into a readily automated, high throughput quantitative technology. Real-time quantitative PCR (qPCR) has become the industry standard for the detection and quantification of nucleic acids for multiple applications, and particularly for the quantification of mRNA expression levels.
    However, a lack of consensus among researchers on how to best perform and interpret qPCR experiments presents a major hurdle for advancement of the technology. This problem is exacerbated by insufficient experimental details in published work, which impedes the ability of others to accurately evaluate or replicate reported results.

  • Steps for a Successful qPCR Experiment
    October 2011  by IDT
    Quantitative PCR (qPCR) is the method of choice for precise quantification of gene expression. qPCR can utilize a variety of probe-based methods such as 5′ nuclease dual-labeled probes, molecular beacons, FRET probes, and Scorpions™ Probes, or use intercalating fluorescent dyes such as SYBR. 5′ nuclease assays have the advantage of the specificity that comes with using a sequence-specific, dual-labeled probe, and is the preferred technique for gene expression analysis. This article will focus on 5′ nuclease assay design and experimental setup considerations that will assist in obtaining accurate and consistent results.
    This article draws upon information published as the MIQE guidelines: Minimum Information for Publication of quantitative Real Time PCR experiments. (2009) Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. Clin Chem. 55(4):611–622.

  • Evolution of Polymerase Chain Reaction - Seminal Technology Continues to Be a Work in Progress
    Feature Articles:   October 1st 2011(Vol. 31, No. 17)  by  Carl T. Wittwer
    Since the discovery of the polymerase chain reaction (PCR) by the oligonucleotide chemist Kary Mullis in 1983, the method has revolutionized molecular biology and clinical diagnostics.
    Before PCR, DNA amplification required multiple steps, including cloning into plasmids, insertion into bacteria, bacterial growth, isolation of plasmid DNA, and separation of inserts from plasmid vectors.
    In contrast, PCR is performed in vitro as a single step, requiring only two oligonucleotide primers, a polymerase, and temperature cycling of the DNA template in the presence of deoxyribonucleotides.
    Although its spread was initially limited by restrictive patent policies, the basic method is now off patent and has become a democratic cornerstone of molecular biology. Thousands of scientists have contributed to and expanded the methods and applications of PCR, including quantification of transcripts after reverse transcription and PCR followed by cycle sequencing.
    Anyone can perform PCR with generic reagents and simple laboratory instruments, amplifying specific DNA segments by 106-to 109-fold for further study in genetics, oncology, and infectious disease.
  • Myth Busted:  A NanoDrop ND-1000 Spectrophotometric reading is insuffiecient to assess RNA quality
    Application Note by Bio-Rad 5893A
  • Assuring Reliability of qPCR & RT-PCR Results - Use of Spectrophotometry on Nucleic Acid Samples Before Experiment Improves Outcome
    20th September 2011,  by Andrew Page & Ilsa Gomez-Curet   in GEN
    The polymerase chain reaction (PCR) is a valuable tool used in both research and molecular diagnostic laboratories because of its specificity, efficiency, fidelity, and relative ease of use.
    Quantitative real-time PCR (qPCR) enables sensitive and accurate quantitative measurement of nucleic acids. Both qPCR and reverse transcriptase PCR (RT-qPCR) are used across a wide range of applications such as gene expression, SNP genotyping, copy-number analysis, pathogen detection, drug target validation, and measurement of RNA interference (RNAi).
    The quality of qPCR and RT-qPCR results can be negatively affected by many experimental variables. To ensure the validity of assay results, sample extraction and preparation steps must be closely monitored, and the starting material must be well characterized before performing RT and qPCR assays.
    Slight differences in pipetting, lack of instrument calibration, improper choice of reference genes, incorrect quantification, and/or use of impure nucleic acid templates can generate erroneous, but believable, results. Therefore, the use of standardized best practices to ensure reliable and meaningful results is recommended. To address the need for standardized qPCR practices, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines have been developed.
  • Welcome to the MIQE website
    August 2011 edited by Stephan Bustin
    The technical standard of most publications utilising qPCR to quantify gene expression is either difficult to judge, since very little information is provided in the "Materials and Methods" sections of most papers, or is poor, since normalisation procedures are inappropriate, most commonly using a single, unvalidated reference gene. The implications of this are disconcerting, since the peer-reviewed scientific literature forms the bedrock of current knowledge and provides the starting point for future experiments.
    The site is an experimental one and its aim is to
        * assemble information relevant to qPCR
        * provide a forum for questions, suggestions and criticisms about qPCR and MIQE
        * review reagents, plastic ware, instruments, meetings
        * initiate discussion about extension of MIQE into areas not currently covered by the guidelines
        * answer questions about experimental protocol, data analysis and publishing requirements
    Over the next few months this web site will be populated with as much impartial information as possible. Any opinions expressed will be those of the (identified) contributor(s) and do not necessarily reflect those of the curator(s) of this web site.
  • Applications of MIQE to Real Time Quantitative PCR
    24th of May 2011- in BioPortfolio - Source: American Biotechnolgist
    Sean Taylor, Field Applications Specialist, Bio-Rad Laboratories, demonstrates how sample quality and reference gene selection effect data analysis and interpretation in real-time quantitative PCR (qPCR) experiments. The presentation is in accordance with the previously published MIQE guidelines. For enhanced viewing, click on the full-screen mode button on the bottom right hand [...]  Original Article: Applications of MIQE to Real Time Quantitative PCR
  • Translate the MIQE guidelines
    Since September 2011 we provide a direct translation of the MIQE guidelines in CHINESE,  JAPANESE,  KOREAN and  RUSSIAN. Please recognize this is an automatic and robotic based translation service, and therefore we provide NO guarantee about the automatic generated content. It should help the world wide qPCR community to understand the core content of the MIQE guidelines.
  • qPCR and MIQE Seminar Series
    Sigma Aldrich Learning Center
    As part of our customer education program, we have provided two recorded seminar series covering the topics of qPCR and MIQE. The recorded sessions are intended to provide a high level overview of these subject matters. We have kept the lessons concise so that you can enjoy a self-paced learning program.
Seminar Title Presenter Recording Length
(hours : minutes : seconds)
Primer and Probe Design Ashley Heath, PhD 0:06:32
An Introduction to qPCR Concepts Mudassir Mohammed, PhD 0:09:37
Selecting a qPCR Basic Detection Chemistry Mudassir Mohammed, PhD 0:12:35
Choosing a Fluorophore / Quencher Combination Anders Bergkvist, PhD 0:11:30
Chemistries for More Challenging qPCR Assays Mudassir Mohammed, PhD 0:15:18
MIQE Concepts Marina Wiklander, PhD 0:03:19
Reference Gene Validation Anders Bergkvist, PhD 0:12:47
Data Analysis Guidelines Anders Bergkvist, PhD 0:10:42



Seminar Title Presenter Recording Length
(hours : minutes : seconds)
MIQE: Assay Design Considerations Tania Nolan, PhD 0:17:37
MIQE: Sample Derived Inhibitors Tania Nolan, PhD 0:13:04
MIQE: RNA Quality Considerations Tania Nolan, PhD 0:15:31
MIQE: RNA Quantity and RT Considerations Tania Nolan, PhD 0:16:38

  • MIQE (Minimum Information for the Publication of Quantitative PCR Experiments) has become the gold standard for assessing the quality and relevance of qPCR-based publications.
    5 August 2011 - Prof Stephen Bustin, Barts and the London School of Medicine and Dentistry


    The real-time polymerase chain reaction uses fluorescent reporter dyes to combine DNA amplification and detection steps in a single tube format. The increase in fluorescent signal recorded during the assay is proportional to the amount of DNA synthesised during each amplification cycle. Individual reactions are characterised by the cycle fraction at which fluorescence first rises above a defined background fluorescence, a parameter previously known as the threshold cycle (Ct) or crossing point (Cp), now standardised by MIQE as the quantification cycle (Cq). Consequently, the lower the Cq, the more abundant the initial target. This correlation permits accurate quantification of target molecules over a wide dynamic range, while retaining the sensitivity and specificity of conventional end-point PCR assays. The homogeneous format eliminates the need for post-amplification manipulation and significantly reduces hands-on time and the risk of contamination. MIQE abbreviates real-time PCR to qPCR, with reverse transcription PCR abbreviated to RT-qPCR.

  • Go, Go Gadgets
    July 13 2011 by Katia Caporiccio
    For researchers working in life science, the MIQE qPCR app for iPhone and iPad, sponsored by Bio-Rad, provides resources and checklists needed to ensure MIQE (Minimum Information for Publication of Quantitative Real-Time qPCR Experiments) compliance for qPCR experiments. “In this day and age, everybody has his phone with him at all times,” said Rachel Scott, senior product manager for Gene Expression at Bio-Rad (Hercules, CA). “The immediacy helps with capturing the information as it occurs, on the fly.”
  • Research gets APP happy
    06/23/2011 by Lisa Grauer
    These two new iPhone and iPad apps promise to advance your research more than any summer intern.
    Bio-Rad’s new MIQE qPCR app also comes equipped with lab tools including extensive qPCR reference information and checklists that allow researchers to ensure MIQE compliance for their qPCR experiments.
    “qPCR is a common lab technique used by most life science researchers today, but not everyone conducting PCR is using the technique in a way that ensures its correct interpretation,” said Rachel Scott, Bio-Rad senior product manager. “As a result of misinterpretation of qPCR data, significant scientific conclusions have been retracted for inaccuracies.”
    Developed by real-time PCR (qPCR) experts Michael W. Pfaffl, professor of molecular physiology at Technische Universität München, and Afif Abdel Nour, associate professor of nurigenomics at the Institut Polytechnique LaSalle Beauvais, the MIQE qPCR app helps researchers achieve accuracy, transparency, and reproducibility in their qPCR experiments by allowing them to monitor MIQE compliance via color-coded checklists and progress bars. The app also provides expert advice through direct links to MIQE-related publications and email addresses of qPCR experts.
  • Saving lives one iPhone at a time
    Monday, July 25th, 2011 by www.americanbiotechnologist.com
    My iPhone is very precious to me. Until I had an iPhone, I wasn’t aware of how much I was missing. Now that I am an iPhone owner, I don’t know how I ever lived without one.
    There are tons of awesome apps out there. For molecular biologists there is the NCBI Blast app, the MIQE app and the qPCR app (among others). There are also a number of cool health apps such as the urine blood glucose monitor or STD detector app.
    Now another cool app has been added to your iPhone’s medical repitoire. The Melenoma Risk Assessment Tool by Health Discovery Corporation, is designed to help users learn about melanoma and identify areas on their skin which may need attention from a physician specializing in the diagnosis of melanoma.
    Using the iPhone camera feature, users can take a picture of their skin lesions and moles and within seconds receive a risk analysis of their uploaded picture being a melanoma. Utilizing your iPhone GPS, MelApp can refer you to a nearby physician specializing in the diagnosis and treatment of melanoma for proper medical follow up, without the need to input a zip code or any personal information. These pictures also can be stored on MelApp and reviewed for changes in the skin lesions occurring over time.
  • The MIQE iPhone App
    Monday, July 25th, 2011 by www.americanbiotechnologist.com
    We have written many posts about the MIQE real time PCR standards that are basic requirements for anyone engaged in real time PCR experimets. Now there is a new tool for all ipod/iphone users to add to their arsenal. A MIQE qPCR app!
    The MIQE app helps scientitst review scientific work and check their own project’s MIQE compliance. Plus, the app includes a list of the most current qPCR news and events and “emergency” contact numbers that you can call/email should you have any questions about your qPCR experiments.
    The application was developed by Dr. Afif Abdel Nour, Associate Professor in Nutrigenomics at LaSalle Beauvais, in collaboration with Dr. Michael Pfaffl and was sponsored by Bio-Rad Laboratories.
  • Implementation of MIQE guidelines to the StellARray system
    July 2011by Lonza AG
    The increasing number of citations of the MIQE guidelines (minimum information for publication of quantitative real-time PCR experiments, (Bustin et al. 2009) demonstrates a growing emphasis on standardized experimental practice for qPCR.
    The SYBR-based StellARray qPCR Array system from Lonza offers a simple and reliable system for gene expression analysis that meets the MIQE standards and makes it easier for qPCR users to compare and publish their results.
    download PDF



qPCR  -  quicker and easier but don't be sloppy

Monya Baker
Nature Methods  8, 207–212 (2011)

Gene profiling using quantitative PCR is becoming higher throughput, but researchers must be careful in gathering their data.


  • MIQE guidelines for future publications on qPCR
    By Dr. Marcus Neusser, European Product Manager Gene Expression, Bio-Rad Laboratories
    In 2009 Stephen Bustin, together with some renowned scientists - all experts on quantitative real-time PCR (qPCR) - published a paper in Clinical Chemistry recommending a set of guidelines with some essential (59) and some desirable (28) check points for documentation of the minimum information necessary for evaluation of qPCR experiments (MIQE) 1. The paper emphasises guidelines to encourage better experimental practice, allowing more reliable and unequivocal interpretation of quantitative PCR results.

  • Video Tutorial: MIQE and Your qPCR Data
    In this video, Dr. Sean Taylor, Field Applications Specialist, Bio-Rad Laboratories, demonstrates how sample quality and reference gene selection effect data analysis and interpretation in real-time quantitative PCR (qPCR) experiments. The presentation is in accordance with the previously published MIQE guidelines.
    For enhanced viewing, click on the full-screen mode button on the bottom right hand corner of the video.

  • Evaluierung der qPCR - Die Real-Time-RT-PCR-Datenanalyse im Fokus der MIQE-Richtlinie
    BIOspektrum May 2011
    MICHAEL W. PFAFFL & IRMGARD RIEDMAIER
    LEHRSTUHL FÜR PHYSIOLOGIE, WISSENSCHAFTSZENTRUM WEIHENSTEPHAN FÜR ERNÄHRUNG, LANDNUTZUNG & UMWELT, TU MÜNCHEN
    Die MIQE-Richtlinie wurde 2009 von einer Gruppe internationaler Wissenschaftler ins Leben gerufen, um die Qualität, die Richtigkeit sowie die Zuverlässigkeit der gewonnenen qPCR-Ergebnisse im Labor und in der wissenschaftlichen Literatur zu steigern.
    The MIQE guidelines were established 2009 by a group of international scientist to improve the quality, the accuracy, and the reliability of the generated quantitative PCR results in the lab and in the scientific literature.

  • Standardisation and reporting for nucleic acid quantification
    March 2011 by 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.

  • Get started with microRNA qPCR
    March 2011
    Exiqon has released a tech note explaining all the basics of setting up a microRNA qPCR experiment. Learn how to choose controls, how to determine the number of replicas, how to analyze the results and much, much more. Read the tech note if you are just getting started with microRNA qPCR or if you are an experienced user looking for tips and tricks.

  • Primer Sequence Disclosure: A Clarification of the MIQE Guidelines
    Stephen A. Bustin, Vladimir Benes, Jeremy A. Garson, Jan Hellemans, Jim Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W. Pfaffl, Gregory L. Shipley, Jo Vandesompele, and Carl T. Wittwer
    Clin Chem published March 18, 2011

  • Bio-Rad's New CFX Manager™ Software 2.0 Streamlines Real-Time PCR Experiment Setup, Data Analysis, and MIQE Compliance
    Hercules, CA — March 16, 2011 — Bio-Rad Laboratories, Inc. introduces new real-time PCR experiment setup and data analysis software, CFX Manager software 2.0, for use with Bio-Rad's CFX96™, CFX384™, and MiniOpticon™ real-time PCR detection systems. From scheduling the use of an instrument to expediting manuscript acceptance, CFX Manager software 2.0 makes running qPCR experiments easier than ever.
    Combined with Biogazelle's qbasePLUS software, which is included with the CFX96 and CFX384 systems, CFX Manager software 2.0 enhances researchers' ability to comply with the emerging best practices standard Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE). Adherence to MIQE is recommended for manuscript submission by publications such as Nucleic Acids Research, Clinical Chemistry, BioMed Central, and BMC Molecular Biology. CFX Manager software 2.0's terminology is consistent with MIQE guidelines (for example, Cq instead of Ct) and data can be exported in the recommended RDML file format for submission with a manuscript or for import into qbasePLUS software.
    CFX Manager software 2.0 augments version 1.5 with these additional key benefits:
    • Stay organized — reserve instrument access using the Scheduler
    • Streamline experiment setup — rapidly prepare reactions using the Master Mix Calculator
    • Make faster decisions about data — easily visualize all run data important to you with Custom Data View
    • Export only the data you need — specify what items to export and in what format with Custom Data Export
    • Quickly integrate with any Laboratory Information Management (LIMS) system — import a plate and protocol template created by LIMS and generate a customized data file ready for LIMS retrieval
    • For more information about CFX Manager software 2.0 => http://bit.ly/fMkAh8

  • MIQE Guidelines - a brief overview
    Posted by cjbornarth@life on Mar 9, 2011 8:00:39 AM
    MIQE is an acronym for “Minimal Information for Publication of Quantitative Real-Time PCR Experiments”. These guidelines are a short list of details, or experimental information, agreed upon by some of the leading scientists in the qPCR field for the benefit of all researchers. When scientists publish using these guidelines, the work will have more credibility in the field, and allow for easier comparison between studies.
  • Nucleic Acids Research - GENERAL POLICIES OF THE JOURNAL
    Authors' responsibilities
    Quantitative PCR - Authors are encouraged to follow the 'Minimal Information for Publication of Quantitative Real-Time PCR Experiments' (MIQE) guidelines, if appropriate. The guidelines are published by the Real-Time PCR Data Markup Language Consortium and can be found at http://www.rdml.org/miqe.php
    Microarray data - All authors must comply with the 'Minimal Information About a Microarray Experiment' (MIAME) guidelines published by the Microarray Gene Expression Data Society, which can be found at http://www.mged.org/Workgroups/MIAME/miame_checklist.html. NAR also requires submission of microarray data to the GEO (http://www.ncbi.nlm.nih.gov/geo/) or ArrayExpress (http://www.ebi.ac.uk/arrayexpress/) databases, with accession numbers at or before acceptance for publication.
  • Quality Control Guidelines - The Real-time PCR Research and Diagnostics Core Facility
    The Real-time PCR Research and Diagnostics Core Facility adheres to the highest quality standards to ensure accurate results. For an outline of our current quality control guidelines, please read the following manual => Real-time PCR Research and Diagnostics Core Facility Quality Control
    Dr. Emir Hodzic's presentation on guidelines to provide authors, reviewers and editors specifications for the minimum information that must be reported for a qPCR experiment in order to ensure its relevance, accuracy, correct interpretation and repeatability.
    • From designing to publishing your data - by Emir Hodzic
      Real-time PCR Molecular & Diagnostic Core Facility,   UC Davis, USA
      Quantitative real-time PCR (qPCR) is a technique that is now commonly employed in almost all molecular biology laboratories to elucidate variation in gene expression. But with the widespread use of such a wonderful and sensitive technology comes differences in how to obtain valuable and reportable results. The lack of quality control for publishing qPCR data is still lacking. To overcome this increasing problem of lack of consistency in publications, a panel of real-time PCR experts published a set of guidelines containing what they consider the minimal information required when reporting qPCR results. The Real-time PCR Research and Diagnostic Core Facility at UC Davis fully abides with the proposed MIQE guidelines, so this presentation presents an expanded explanation of the guideline items with commentary, based on our experience, on how those requirements might be met prior to publication.
  • MIQE guidelines for future publications on qPCR
    27 January 2011 - by Dr. Marcus Neusser, European Product Manager Gene Expression, Bio-Rad Laboratories
    In 2009 Stephen Bustin, together with some renowned scientists - all experts on quantitative real-time PCR (qPCR) - published a paper in Clinical Chemistry recommending a set of guidelines with some essential (59) and some desirable (28) check points for documentation of the minimum information necessary for evaluation of qPCR experiments (MIQE) 1. The paper emphasises guidelines to encourage better experimental practice, allowing more reliable and unequivocal interpretation of quantitative PCR results.
  • MIqPCR - MIQE - RDML -  slideshow by Andreas Untergasser
    Intersting but short slide show explaing the evolution from MIqPCR to trhe MIQE guideleines and the importance of the RDML script.
  • Chapter 8 - The MIQE Guidelines Uncloaked
    Gregory L. Shipley
    Publication date - January 2011
    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.
    in   PCR Troubleshooting and Optimization:    The Essential Guide,   ISBN: 978-1-904455-72-1
    Publisher: Caister Academic Press
    Editors: Suzanne Kennedy and Nick Oswald MO BIO Laboratories, Inc., Carlsbad, CA 92010, USA and BitesizeBio, Edinburgh, UK
  • The MIQE Guidelines and Assessment of Nucleic Acids Prior to qPCR and RT-qPCR
    Andrew F. Page
    ,  Thermo Fisher Scientific - NanoDrop products Wilmington, Delaware USA
    APPLICATION NOTE  -  NanoDrop Spectrophotometers
  • Solaris qPCR-A breakthrough in qPCR probe-based specificity
    March 2011 - Kirsteen H. Maclean PhD, Thermo Scientific
    Today the use of real-time quantitative PCR (qPCR) is ubiquitous in almost every research laboratory for quantification of gene expression. Current detection strategies are based on an increase in fluorescence either from the use of double-stranded intercalating dyes (SYBR GreenTM) or probe based assays (e.g hydrolysis or separation probes) which allow the end user to assess proportional increases of target. The fluorescence is monitored during each cycle of PCR by way of the now familiar amplification plot. Unfortunately, while the PCR process itself is theoretically simplistic, there exists a lack of consensus within the scientific community with respect to experimental design, data reporting and analysis for qPCR strategies. In an effort to provide standardization when reporting qPCR results, key opinion leaders in the PCR community published a set of guidelines known as “The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE)” (1).  The aim of this publication is to provide a benchmark for the quality assessment of a qPCR assays reported in a given publication. The MIQE guidelines now define the minimum information required for evaluation of qPCR results, and include a checklist to be included in the initial submission of a manuscript to a publisher. Concordantly, the Thermo Scientific Solaris qPCR Gene Expression Assays are a novel type of primer/MGB-probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/MGB-probe sets are pre-designed feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts. Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting up the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the MGB-probe and primer sequence information are provided, simplifying the publication process to be MIQE compliant. While Solaris qPCR was only released within the last year; several research groups have quickly embraced the advantages of this novel MGB-probe-based technology to address their specific scientific needs. Recent publications, two described herein citing the utility of Solaris qPCR gene expression assays highlight the significance of this new streamlined technology for specific target quantification.
  • UPDATE - Life Technologies’ TaqMan® Assays QPCR Guarantee Program Sets New Industry Standard for Customer Service and Support
    Life Technologies launched the TaqMan® Assays QPCR Guarantee Program, which is designed to provide customers with unparalleled peace of mind by offering to replace any of our more than 7 million pre-designed TaqMan® assays that don’t meet their expectations (see QPCR video).
    The program helps take some of the risk out of their research by guaranteeing the quality, performance, content, and results (hence the acronym “QPCR”), across Life Technologies’ line of pre-designed TaqMan® Assays. If for any reason a pre-designed assay does not perform to the level of our customers’ satisfaction, Life Technologies will first help troubleshoot the issue and, if unsuccessful, we will replace the assay or credit their account.
    This is Life Technologies’ way of formalizing our commitment to our customers by standing behind the industry’s best-performing and most-consistent pre-designed qPCR assays.
    TaqMan® pre-designed assays are used in laboratories around the world among clinical, pharmaceutical, agricultural and academic researchers.  They serve as powerful tools to rapidly and precisely measure genomic and proteomic changes in studies that are applied toward disease research, drug discovery, and agricultural development.
    Certain restrictions apply. For details and complete terms and conditions regarding the TaqMan® Assays QPCR Guarantee, please visit www.appliedbiosystems.com/taqmanguarantee

    Advanced qPCR Techniques for Publication Success:   Following MIQE Recommendation
    Overview -The real-time reverse transcription (RT) polymerase chain reaction (PCR) (RT-qPCR) and real time PCR methods address the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology, diagnostics and other areas and have become the methods of choice for the quantification of nucleic acid targets and identification of sequence specific variations. Although often described as a “gold” standard, these are far from being routine assays.
    Date Location Register Agenda
    July  11–15, 2011 EMBL,  Heidelberg,  Germany Register now Download  (194 Kb PDF)

2010
  • 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
  • The Importance of Quality Control During qPCR Data Analysis
    Barbara D’haene, Ph.D. & Jan Hellemans, Ph.D. Biogazelle & Ghent University
    International Drug Discovery 2010
    Since its introduction in 1993, qPCR has paved its way towards one of the most popular techniques in modern molecular biology [1]. Despite its apparent simplicity, which makes qPCR such an attractive technology for many researchers, final results are often compromised due to unsound experimental design, a lack of quality control, improper data analysis, or a combination of these. To address the concerns that have been raised about the quality of published qPCR-based research, specialists in the qPCR field have introduced the MIQE guidelines for publication of qPCR-based results [2]. The main purpose of this initiative is to make qPCRbased research transparent, but the MIQE guidelines may also serve as a practical framework to obtain high-quality results. Within the guidelines, quality control at each step of the qPCR workflow, from experimental design to data analysis, is brought to the attention as a necessity to ensure trustworthy results.....
  • The Marketing of Science
    December 3, 2010
    I am a scientist for profit. This means, as you are well aware, I have to work with marketing people to generate pretty pictures showing perfect results with any product that we sell. You know those flyers and brochures and ads in BioTechniques where a tiny picture of a gel or a qPCR assay with photoshop perfect curves or bands is plopped on the page next to some meaningless picture and supposed to convince you to call or go to a website? Those things.
    Before working for a company, I would take a look at those pictures but I never put much stock into them. I mean, of course they're going to show perfect data. What else will they show? Their kit sucks next to a competitor? So marketing data never really did sway me much. I looked at it, but not in any depth. I guess, I expect there to be some attempt at science in the ad, but it's merely representative data.
    My first biotech job wasn't in marketing.  The company I worked for was and still is considered one of the best in the world and I was so very proud to be a part of that company. When they would introduce a new product, the product manager would come present all the beautiful R&D data proving the product works and it was convincing. I would walk away from those meetings absolutely positive that this was the best damn invention in the world and we have geniuses in R&D and how lucky am I to represent such brilliance.
    About this first company, I still do believe that they have geniuses in R&D. However, since leaving, I feel that their employees are extremely self-obsessed and self-absorbed but I can understand why they are that way. It is part of the company culture. But that isn't the point of this article......... =>  read more

  • The Story of MIQE and its Impact for Future Publications on qPCR
    Questions to an Expert in qPCR, Stephen Bustin (Ph.D.)  -  The Story of MIQE and its Impact for Future Publications on qPCR

    November 2010
    MIQE is a set of guidelines with some essential (59) and some desirable (28) check points for the documentation that describes the minimum information necessary for evaluation of quantitative real-time polymerase chain reaction experiments. Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of quantitative PCR results. More details can be found on Stephen Bustin’s MIQE homepage: http://www.sabustin.org/
  • MIQE – Minimum Information for Publication of Quantitative Real-Time PCR Experiments
    suppoted by Sigma-Aldrich
    The potential applications for Quantitative Real-Time PCR (qPCR) have increased exponentially since the first description (Higuchi, 1993). However, researchers have been frustrated by complications such as contamination, insufficient amplification, low sensitivity, and uncertainty about what constitutes a suitable statistical analysis. Until recently, there has been a lack of consensus about how to deal with these obstacles.
    An international research team, including Dr. Tania Nolan, Sigma's Global Manager for Applications and Technical Support, published The MIQE (pronounced Mykee) Guidelines in 2009 to address the challenges of performing dependable qPCR measurements. By following MIQE, you are certain to produce more reliable data and will:
        * Promote experimental transparency
        * Ensure consistency between laboratories
        * Maintain the integrity of the scientific literature
    Sigma's qPCR services, including primer and probe designs, assay protocol development, troubleshooting, and data analysis support, adhere to MIQE, which allows you to publish or bring your product to market faster and with confidence.   =>  read more
Blitzlicht  MIQE-Richtlinien
Qualität und Richtigkeit von qPCR-Ergebnissen steigern
Laborwelt December 2010
by Michael W. Pfaffl, TUM, Freising-Weihenstephan

Die Anwendung der quantitativen Polymerase-Kettenreaktion (qPCR) oder die Kombination der qPCR mit der reversen Transkription (RT) ist zu einem Routinewerkzeug in der modernen mole-kularbiologischen Forschung und molekularen Diagnostik geworden. Das Expression Profiling biologischer Proben auf mRNA- und microRNA-Ebene mittels quantitativer RT-PCR (RT-qPCR) ist von großem Nutzen und liefert wichtige Ergebnisse in zahlreichen biologischen Disziplinen. Vor allem in der Routinediagnostik, der universitären und industriellen Forschung sowie in der funktionellen Genomforschung ist sie unverzichtbar.
Full issue - Laborwelt - PCR Spezial - Dezember 2010

  • A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines.
    Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M.
    Bio-Rad Laboratories, Inc., Hercules, CA 94547, USA.
    in   -  The ongoing Evolution of qPCR   -  Methods. 2010 Apr;50(4): S1-5.  http://evolution.gene-quantification.info
    Given the highly dynamic nature of mRNA transcription and the potential variables introduced in sample handling and in the downstream processing steps (Garson et al. (2009)), a standardized approach to each step of the RT-qPCR workflow is critical for reliable and reproducible results. The MIQE provides this approach with a checklist that contains 85 parameters to assure quality results that will meet the acceptance criteria of any journal (Bustin et al. (2009)). In this paper we demonstrate how to apply the MIQE guidelines (www.rdml.org/miqe) to establish a solid experimental approach.

  • IDT publishes free downloadable qPCR user guide
    8 December 2010
    User guide provides a MIQE compliant overview of this essential research technique!
    Integrated DNA Technologies has developed an extensive quantitative real-time polymerase chain reaction (qPCR) user guide, which is available as a free download.
    The manual provides user guidance on the entire qPCR process - from RNA isolation to data analysis -  covering the basics of experimental set-up, performance and analysis. Specific information on 5’ nuclease assays, including re-suspensions and qPCR protocols are also supplied, as well as a troubleshooting section which discusses commonly encountered issues. The document is written in compliance with MIQE guidelines: minimum information for publication of quantitative real-time PCR*.   www.idtdna.com

    *Bustin et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem, 2009 55(4): 611-622
  • Life Technologies’ TaqMan® Assays QPCR Guarantee Program Sets New Industry Standard for Customer Service and Support
    by Sam Raha, Vice President and General Manager of Genomic Assays  19 November 2010
    Today we are launching Life Technologies’ TaqMan® Assays QPCR Guarantee Program, which is designed to provide customers with unparalleled peace of mind by offering to replace any of our more than 7 million pre-designed TaqMan® assays that don’t meet their expectations (see QPCR video).
    The program helps take some of the risk out of their research by guaranteeing the quality, performance, content, and results (hence the acronym “QPCR”), across Life Technologies’ line of pre-designed TaqMan® Assays. If for any reason a pre-designed assay does not perform to the level of our customers’ satisfaction, Life Technologies will first help troubleshoot the issue and, if unsuccessful, we will replace the assay or credit their account.
    This is Life Technologies’ way of formalizing our commitment to our customers by standing behind the industry’s best-performing and most-consistent pre-designed qPCR assays.
    TaqMan® pre-designed assays are used in laboratories around the world among clinical, pharmaceutical, agricultural and academic researchers.  They serve as powerful tools to rapidly and precisely measure genomic and proteomic changes in studies that are applied toward disease research, drug discovery, and agricultural development.
    Certain restrictions apply. For details and complete terms and conditions regarding the TaqMan® Assays QPCR Guarantee, please visit www.appliedbiosystems.com/taqmanguarantee
  • Gene Expression Assay Performance Guaranteed With the TaqMan® Assays QPCR Guarantee Program
    Real-time or quantitative PCR (qPCR) is one of the most powerful and sensitive techniques available for gene expression analysis. It is used for a broad range of applications, including quantification of gene expression, measuring RNA interference, biomarker discovery, pathogen detection, and drug target validation. When studying gene expression with qPCR, scientists usually investigate changes—increases or decreases—in the quantity of particular gene products or a set of gene products. Investigations typically evaluate gene response to biological conditions such as disease states, exposure to pathogens or chemical compounds, the organ or tissue location, or cell cycle or differentiation status.
  • Publishing Data That Conform to the MIQE Guidelines
    Minimum information for publication of Quantitative Real-Time PCR
    Experiments (MIQE) guidelines help researchers design qPCR experiments.
  • Are you MIQE compliant?
    2010 by Premier Biosoft International
    Since its introduction, real-time PCR has become the main technical platform for nucleic acid detection in research and development. This technology has become an invaluable tool for many scientists working in different disciplines. Especially in the field of molecular diagnostics, real-time PCR - based assays have gained favor in the recent past. Although many significant results have been derived from real time PCR studies, one limitation has been the lack of standards to perform and interpret these experiments.
    The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines outline the minimum information that should be included while describing a real time PCR experiment, to standardize the results, to easily interpret them and to independently verify them. MIQE guidelines have been written to promote consistency between laboratories and increase experimental transparency.
    Download the MIQE Checklist
    Is your experiment MIQE Compliant?

    AlleleID® and Beacon Designer™, our Real Time PCR oligo design software provide everything a researcher needs to meet MIQE compliance, making submission for publication review more efficient.
  • Making the most of MIQE
    BMC Molecular Biology - October 2010
    The Editorial Board of BMC Molecular Biology endorse a new set of essential MIQE-light guidelines for the reporting of quantitative PCR data: "MIQE precis", and provide guidance for the suitability of pure reference gene papers to the journal.
  • QC Best Practices for the qPCR Lab
    Live Event:   Thursday, July 29, 2010 at 2:00 PM EDT
    Moderator:    Robert Fee, Editor-in-Chief , Bioscience Technology   
    Panelist:       Manju Sethi, Senior Product Manager, Thermo Fisher Scientific

MIQE precis: 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 precis: with reference to reference genes
BioMed Central Blog - Sep 21, 2010
Genes that maintain constant expression under a variety of circumstances are known as ‘reference genes’. They are vital for researchers who need to quantify gene expression changes in other genes and need a ‘reference point’ against which to do so. BMC Molecular Biology, has to date published around 200 reference gene-related papers from researchers working in such diverse models as peaches, sharks, barnacles and glioblastoma to name but a few.
However, to be a true reference gene you need to fulfil a certain list of criteria and the research field is now united in requesting that all work be performed to the same accuracy and in accordance with recommended guidelines. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines were launched over a year ago by an international team of researchers. The aim of these guidelines was to enable the benchmark technology for measuring gene expression (quantitative PCR [qPCR]) to become standardised when reported in research papers. The MIQE guidelines advise on good assay design and appropriate data analyses for nucleic acid detection and quantification. BioMed Central supports and promotes initiatives aimed at improving the reporting of biomedical research, and refers authors  to the MIBBI Portal (of which MIQE is part of) for reporting biological and biomedical research. Whilst some authors have included MIQE checklists as supplemental files with their work (for example here), there has been some debate as to the utility and ease in doing this in all cases.
After working with several Editorial Board Members from BMC Molecular Biology, we propose that all researchers wishing to publish qPCR work do so by adhering to our simpler and more abridged 'light' guidelines – MIQE précis. We also propose that the majority of reference gene papers are no longer suitable for publication as ‘pure reference gene papers’, but this information will need to be incorporated as part of a larger study. Alternatively, authors may publish these more incremental (but still potentially useful) pure reference gene articles in BMC Research Notes to contribute to our topical series: “Quantitative Real Time PCR normalization and optimization”



  • The Future of qPCR: Best practices, Standardization, and the MIQE Guidelines
    September 30, 2010 - 12 noon Eastern, 9 a.m. Pacific, 4 p.m. GMT
    Quantitative polymerase chain reaction (qPCR) has emerged as a powerful tool in molecular biology laboratories, both in research and in diagnostic settings. Even as qPCR grows in popularity, it is being recognized that there are some challenges associated with the technology, particularly with respect to reproducibility within and between laboratories. Fortunately, many of these limitations can be addressed through a standardized set of best practices. Using the recently published MIQE guidelines as a foundation, our expert panel will address the best practices of qPCR, with the goal of providing researchers with more consistent and reliable data.
    register  
    During the webinar, the panelists will:
    • provide an overview of the MIQE guidelines
    • address qPCR applications and primary challenges
    • outline best practices and assay design to get the best out of your qPCR
    • describe the essential quality control steps, including nucleic acid quantification
    • answer your questions during the live Q&A session.
    Participants:
    •       Stephen A. Bustin, Ph.D.;  Queen Mary, University of London;  London, UK
    •       Gregory L. Shipley, Ph.D.;  University of Texas Health Science Center at Houston;  Houston, TX
    •       Manju R. Sethi;  Thermo Fisher Scientific;  Wilmington, DE

MIQE Guidelines slowly entering "high impact" journals!

Routine lab method's accuracy called into question
Catherine Shaffer
Nature Medicine Vol 16, page 349 (2010)

download PDF                 link to Nature Medicine
PCR proponents =>
Stephen Bustin (left) & PCR inventor Kary Mullis


In 2002, four years after first sparking public controversy over whether the measles, mumps and rubella vaccine causes autism, Andrew Wakefield reported a possible molecular mechanism for the connection. He claimed that a form of irritable bowel disease, which he called autistic enterocolitis, was triggered by the measles virus (Molec. Pathol. 55, 84–90, 2002). That finding, however, was based on a “defective experimental technique,” Stephen Bustin, a molecular biologist at Barts and the London School of Medicine and Dentistry, told a US federal court in 2007. The problem: Wakefield had incorrectly applied the common laboratory protocol known as quantitative real-time polymerase chain reaction (qPCR) to come to his conclusions.
Bustin says this faulty lab work is a problem shared by many researchers around the world who have turned to qPCR to measure gene expression. Unlike standard PCR, which can only crudely quantify levels of DNA, the chemistry behind qPCR allows researchers to assess such levels more precisely by comparing sequences of interest against a known reference added to the test tube mix as a control.
But the reference genes used in qPCR can vary between experiments and laboratories, which can give misleading results or make it difficult to compare one study to another. As a result of this and other variables in the technique, a majority of scientific papers involving qPCR include flawed methods, say a team of leading qPCR experts. Most qPCR methods, as reported in the literature, are improperly validated and irreproducible, Bustin claims.
Last year, he and 11 colleagues published a set of more than 60 individual standards - collectively called the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE)  to address this problem  ( Clin. Chem. 55, 611–622, 2009 ).
“If you look at the literature, you find again and again and again the appalling quality of qPCR protocols,” says Bustin, who this month repeated his call for the scientific community to adopt the MIQE guidelines (Methods 50, 217–226, 2010). “There's no excuse for anyone either not reporting or not doing experiments properly.”
The consequence of poor methodology is that many published papers contain erroneous conclusions, says Mikael Kubista, a coauthor of the MIQE guidelines and chief executive of the TATAA Biocenter in Göteborg, Sweden. “The problem is that the technique itself seems so simple and so easy to do, [but] in real life you're analyzing biological samples with complexity.”
Wakefield's 2002 study reported the presence of measles virus in the gut, yet the authors hadn't included a reverse-transcription step to convert the RNA virus into DNA in some of their qPCR runs, and so they probably detected a DNA contaminant, according to Bustin's testimony (Eur. Pharm. Rev. Dig. 1, 11–16, 2008). This error and others like it could be prevented by following correct methodology, Bustin says. The MIQE guidelines, for example, call for a detailed description of the reagents used in the technique, including the enzyme type used for the RNA reverse transcriptase step. (A follow-up publication that included the 2002 paper's corresponding author John O'Leary, of Trinity College Dublin, among others, used the same methods as the original study and found no link between measles and autism (PLoS One 3, e3140, 2008).   (Neither Wakefield nor O'Leary was available for comment.)
Not all researchers are convinced that the MIQE guidelines are the perfect solution. “There's no doubt that there is a need for improved standardization,” says Helen Fernandes, director of molecular diagnostics at the University of Medicine and Dentistry of New Jersey in Newark, who is helping evaluate protocols for the Clinical and Laboratory Standards Institute, a global organization supporting consensus lab procedures. But “we have to consider other views or other guidelines, as well,” she explains.
Many researchers might be reluctant to adopt the guidelines unless major journals first change their publication policies. Journal editors, however, are hesitant to impose new rules without a broader scientific consensus. “We would be delighted to embrace the [MIQE] guidelines, but we are not really persuaded that the guidelines are embraced by the community,” says Juan Carlos López, editor-in-chief of Nature Medicine, which does not require that authors adhere to MIQE. This view is reflected in the policies of most leading journals, including Cell, Science, Nature, PLoS, New England Journal of Medicine and The Lancet, which do not mention qPCR data in their instructions for authors, although many have instructions for other common lab techniques such as DNA microarrays.
One publisher that has warmed to MIQE is London-based BioMed Central (BMC). Although adherence to the principles is not explicitly required of authors, BMC journal editors and reviewers use them to guide disputes over how qPCR data should be reported. “Where there has been methodological information lacking or issues raised about the quality of these particular experiments, it has been extremely useful to quote the MIQE guidelines,” says BMC's senior scientific editor Scott Edmunds. However, BMC has no plans make the guidelines compulsory, he adds.

  • Meeting Report - Developments in real-time PCR research and molecular diagnostics
    Stephen A Bustin
    Expert Review of Molecular Diagnostics
    September 2010, Vol. 10, No. 6, Pages 713-715
    This meeting was designed to highlight the wide range of new methods, instruments and applications that underlie the popularity of quantitative real-time PCR technology in all areas of life science research, as well as in clinical diagnostics. It provided a fascinating snapshot of current trends and novel approaches, as well as important issues concerning assay design, optimization and quality control issues.

  • A Practical Approach to MIQE for the Bench Scientist
    In a groundbreaking review published in February 2009, Bustin et al bemoaned the lack of standardization in Quantitative Real-Time PCR (qPCR) experimentation and data analysis. In their critique the authors cite the use of diverse reagents, protocols, analysis methods and reporting formats which has negatively impacted on the acceptance of qPCR as a robust quantitative molecular tool.
  • Illumina ecoqpcr Software
    The Eco Real-Time PCR software interfaceEvery Eco system includes a Netbook computer pre-installed with flexible, easy-to-use software that integrates user control, real-time data collection, and advanced data analysis. The software conforms to MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines, making data analysis and submission for publication review more efficient.
    - paper MIQE guidelines
    - MIQE guideline checklist
    Eco software uses a unique icon-driven user interface to simplify experimental design and setup. Pre-set defaults for plate setup and thermal profile are provided for the most commonly used experimental protocols. Temperature and time for each protocol step can easily be changed by click-and-drag action with the mouse. Experiment templates can be customized and saved for future use. All qPCR chemistries and  all standard Real-Time PCR applications are supported, with High Resolution Melt (HRM) analysis as a standard option.

Efforts to standardize qPCR data meets mixed reviews

05/25/2010
Uduak Grace Thomas
BioTechniques

One year ago, an international team of researchers proposed guidelines for the publication of real-time PCR experiments. Since then, there has been mixed response from the scientific community. Uduak Thomas investigates the reasons behind the resistance.

  • qPCR's Big Bag of Tricks - June 2010 by Tracy Vence
    Assuring quality

    But as qPCR has been gaining attention for its applications that have identified new targets, it hasn't been kept properly in check, says Stephen Bustin, a professor of molecular science at the Queen Mary University of London. "This attention is accompanied by a reluctance to question the reliability and relevance of the qPCR data," Bustin says. "Unlike many diagnostic assays it is threatening to replace," he continues, "qPCR is not a mature technology — there are serious disagreements on how best to perform the assay, how to obtain copy numbers or relative quantification data from raw quantification cycles, and whether linear regression or non-linear regression algorithms are most suitable for data analysis."
    In a 2009 Clinical Chemistry paper, Bustin and other PCR experts suggested the minimum information for publication of quantitative real-time experiments, or MIQE. Because Bustin et al. note that "full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results," they argue that MIQE details should accompany every peer-reviewed qPCR paper, whether in an abbreviated form or as a supplement.
    Bustin predicts that there will be "continued publication of contradictory results, persistence of uncertainty, and, consequently, lack of confidence by clinicians in PCR data as the basis for their diagnostic and prognostic decision-making" until there is a community-wide agreement on how to best standardize the technology.
    Ghent's D'haene says a "big challenge remains [in the] adherence of qPCR-based scientific articles to the recently published MIQE guidelines." In abiding by standardized practices, she says, studies will become "much more transparent, reproducibly in other labs, and simply lead to higher-quality and trustworthy conclusions."

  • The MIQE Guidelines Uncloaked - Speaker: Greg Shipley
    8 June 2010
    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 presentation presents an expanded explanation of the guideline items with commentary on how those requirements might be met prior to publication.
  • Do Your RT-qPCRs Make The Grade?  -  Tech Tip
    by Suzanne Kennedy
    Real-time PCR is a technique that is now commonly employed in almost all molecular biology laboratories to quickly answer very specific questions. Northern and Southern blotting are now a thing of the past. No longer do we wait days to know whether a gene is expressed. We can have the answer in 45 minutes!
    But with the widespread use of such a wonderful and sensitive technology comes differences in how results are reported in the literature. There are also differences between reviewers reading these papers and their understanding of the essential information required to judge the accuracy of the reported data.
    To overcome this increasing problem of lack of consistency in publications, a panel of real-time PCR experts published a set of guidelines containing what they consider the minimal information required when reporting qPCR results. That paper called The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, was published February 2009 in the Journal of Clinical Chemistry.
  • Comply with MIQE guidelines for qPCR  -  Agilent 2100 Bioanalyzer assessment of RNA integrity
    by Ruediger Salowsky - Agilent Product Manager Bioanalyzer - RNA/DNA Solutions
    Quantitative real-time polymerase chain reaction (qPCR) and microarray analysis have become essential for elucidating variations in gene expression. While guidelines that define the minimum information required for interpretation of microarray data have been available since 2001,[1] similar specifications for qPCR experiments have been developed only recently. In early 2009, a consortium of leading scientists who use qPCR, established specifications for the minimum information that you must report for a qPCR experiment that you wish to publish. These are the MIQE guidelines (for minimum information for publication of quantitative real-time PCR experiments). This article describes how the Agilent 2100 Bioanalyzer helps you meet these requirements.
  • The MIQE Guidelines - Minimum Information for Publication of Quantitative Real-Time PCR Experiments
    initiative by PrimerDesign
    During the past decade, several high-profile cases of faulty research have been linked to inconsistent real-time PCR techniques and experiments. In April 2009, Stephen Bustin, a molecular science professor at the school of medicine and dentistry at Queen Mary University of London and an international team of nine scientists, joined forces and developed a set of guidelines for the publishing qPCR results. The resulting 'MIQE guidelines' outline the minimum information required to publish quantitative real-time PCR data with scientific integrity.
    PrimerDesign is cited in the MIQE guidelines because we share the same philosophy on primer sequences as the authors. We have always provided the primer sequences with our custom designed assays as we believe that this is crucial to perform research with integrity.
    We strive to make all of our products compliant with the MIQE guidelines and will always be on hand to guide and advise you in producing real-time PCR data of the highest quality.
  • High-Impact Journals, Large Vendors Contributing to Lack of Quality Control for qPCR Data Publication
    June 03, 2010 - By Ben Butkus
    GÖTEBORG, Sweden – Quality control for publication of quantitative PCR data is still severely lacking – and, in the case of high-profile scientific journals such as Nature and Science, it is "absolutely appalling and scandalous" – according to Stephen Bustin, one of the scientists leading the drive to adopt a set of standards intended to guide high-quality and reproducible qPCR experiments within the field..............................

  • Following MIQE Recommendations - EMBL Heidelberg, Germany
    Monday 5 July - Friday 9 July 2010
    Since the early descriptions of the use of quantitative Real Time PCR, the technique has been adopted in almost every aspect of life science research and is increasingly used for clinical analysis. Over time protocols and strategies have been tried and tested, amended and developed such that there are currently several different approaches. Protocol variations are evident at each step of the RT-qPCR process, from sample acquisition to data analysis (e.g. sample QC, experimental design, assay design and validation, normalisation, biostatistical interpretation, reporting, etc). It is now apparent that these adaptations may result in differences in the final biological conclusion of the study.
    This workshop is based upon the MIQE guidelines. Each step of the RT-qPCR process will be discussed and protocol variations illustrated practically. The student will be instructed in best practice and acceptable alternative strategies.

  • How to design your qPCR experiment so that it conforms to the MIQE guidelines
    There have recently been some high profile retractions of scientific papers that have reported data which turns out to be artifacts of poorly designed experiments.  The MIQE guidelines is a daunting list of 85 items that need to be addressed before a reviewer will accept your qPCR results as valid.
    In the PDF below, the essential elements of sample preparation and experimental design are outlined as a practical guide to meeting the MIQE guidelines.  If you use the approach followed in this guide, you will save countless hours of effort trying to figure everything out on your own.  Sean Taylor, Michael Wakem,  and Greg Dijkman have contributed their many years of qPCR experience, and when you follow this guide, your gene expression experiment will meet the MIQE guidelines.
    Onsite hands-on training for qPCR gene expression studies is available.  Please use the contact form to ask for a quote, and make sure you include your location (city).
    This is a reprint of the article published in Methods: April 2010 - Practical Guide to MIQE guidelines
    Now is a great time to consider an instrument like the Experion to validate the quality of your RNA.  If you do not already have a Bioanalyzer or an Experion, you will need to run your RNA samples out on a gel.

  • Professor calls for urgent change in research methods after Dr Andrew Wakefield is struck off
    Monday 24 May 2010
    Disgraced Dr Andrew Wakefield used research methods which were flawed - but which remain commonplace in the scientific world, according to a professor who gave evidence against him.
    Professor Stephen Bustin, based at Queen Mary University London, was one of the research scientists who gave evidence against Dr Wakefield in 2007, and about the quality of the science he used to prove his now-discredited theory about the MMR vaccine.

  • Nucleic Acid Electrophoresis
    Monday April 12, 2010  by Catherine Shaffer
    Don't have time to pour your own agarose gel, make your own buffers, and wait more than an hour for the results? You're in luck, because nucleic acid electrophoresis is getting easier every day. Using precast gels, all-in-one kits, and even automation, you can make a time-consuming, tedious task as simple as microwaving a bag of popcorn—and get better results than your grandfather did when he was pouring his own gels back in 1990. And although agarose and polyacrylamide gel electrophoresis are reliable, rock-solid techniques that have been in use for decades, there are still some surprising innovations to be made by thinking outside the gel box.
  • A Practical Guide to Publishing RT-qPCR Data That Conform to the MIQE guidelines

    March/April 2010

    Online version in American Biotechnology Laboratory

  • A Practical Guide to Publishing RT-qPCR Data That Conform to the MIQE guidelines
    In an effort to assist the scientific com- munity in producing consistent, high- quality data from qPCR experiments, the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines has been recently published.

  • Documenting Real-Time PCR  -  by Catherine Shaffer, Contributing Editor
    Drug Discovery & Development - April 01, 2010

    In February 2009, twelve internationally recognized experts published a long-awaited set of guidelines for real time PCR experiments after more than a decade of public discussion of how to standardize the method and its reporting. These, guidelines called Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE),1 can be found online at www.rdml.org/miqe.php.
    The spirit of the guidelines is to standardize and streamline the real-time PCR workflow from the early planning stage through publication. Many real-time PCR experiments currently suffer from a lack of standardization and detail in publication. Some of them are flawed by poor experimental design. Although the guidelines are barely a year old, awareness and use of MIQE is not spreading as quickly as many of the thought leaders had hoped. Instrument and reagent vendors are helping out by providing MIQE compliant products and MIQE training resources for customers.
  • Step up to the MIQE    by    Richard Kurtz
    Tuesday, March 30, 2010
    Over the years, polymerase chain reaction (PCR) has evolved into a readily automated, high throughput quantitative technology. Real-time quantitative PCR (qPCR) has become the industry standard for the detection and quantification of nucleic acids for multiple application, including quantification of RNA levels. But a lack of consensus among researchers on how to best perform and interpret qPCR experiments presents a major hurdle for advancement of the technology. This problem is exacerbated by insufficient experimental detail in published work, which impedes the ability of others to accurately evaluate or replicate reported results......
  • Real-time PCR    on    SciTopics
    UPDATE on 21 January 2010 by Prof Stephen Bustin
    Category:  Biochemistry, Genetics and Molecular Biology
    Guidelines for minimum information required for publication of qPCR data have now been published in Clinical Chemistry
  • qPCR Assay Quality Assessment    on    SciTopics
    UPDATE on 21 January 2010 by Prof Stephen Bustin
    Category:  Biochemistry, Genetics and Molecular Biology
    Guidelines for minimum information required for publication of qPCR data have now been published in Clinical Chemistry

2009


  • REVIEW  of the MIQE publication by UHN Mircoarray Centre, Toronto, Canada
    Summary of: Bustin SA, et al. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR
    Experiments. Clinical Chemistry 2009, 55(4):611-622
  • Standardization of qPCR Data Reporting
    by  Kirsteen H. Maclean Ph.D.
    Since its discovery by Kary Mullis and colleagues in 1983, the use of the polymerase chain reaction (PCR) has been a mainstay of scientific research and discovery [1]. Indeed in discussing its inaugural “Molecule of the Year” in 1989, the journal Science provided a concise explanation as to the simplicity of the PCR process:
    "The starting material for PCR, the 'target sequence,' is a gene or segment of DNA. In a matter of hours, this target sequence can be amplified a million fold. The complementary strands of a double-stranded molecule of DNA are separated by heating. Two small pieces of synthetic DNA, each complementing a specific sequence at one end of the target sequence, serve as primers. Each primer binds to its complementary sequence. Polymerases start at each primer and copy the sequence of that strand. Within a short time, exact replicas of the target sequence have been produced. In subsequent cycles, double-stranded molecules of both the original DNA and the copies are separated; primers bind again to complementary sequences and the polymerase replicates them. At the end of many cycles, the pool is greatly enriched in the small pieces of DNA that have the target sequences, and this amplified genetic information is then available for further analysis."
  • Step up to the MIQE    by    Richard Kurtz
    When it comes to real-time PCR in drug discovery, Richard Kurtz believes that MIQE guidelines will help create a clear path to better results. Polymerase chain reaction (PCR) has evolved into a readily automated, high throughput quantitative technology. Real-time quantitative PCR (qPCR) has become the industry standard for the detection and quantification of nucleic acids for multiple applications, and particularly for the quantification of mRNA expression levels. However, a lack of consensus among researchers on how to best perform and interpret qPCR experiments presents a major hurdle for advancement of the technology. This problem is exacerbated by insufficient experimental details in published work, which impedes the ability of others to accurately evaluate or replicate reported results.........
  • Le linee guida MIQE - The MIQE guidelines - talk by Paolo Scaruffi
  • MIQE Guidelines 'Slowly Filtering Through' PCR Community Despite Lack of Journal Enforcement
    December 31, 2009
    Although the guidelines are beginning to catch on among researchers and vendors, they appear to have made little or no impact on the quality of the published literature over the last year.
  • Videos explaining MIQE guidelines
    November 11, 2009
    Browsing through You Tube just now, I found these videos illustrating the concepts of the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. These focus on how to apply the guidelines to design a solid experimental approach for RT-qPCR. There are four videos in total.  The sound is a bit “fuzzy,” but the content is a fairly nice overview of MIQE.
  • Helixis Tutorial: MIQE guidelines: a bench perspective on use and benefits
    Description: "MIQE guidelines from a scientist perspective and discussion on their use and benefits when performing Real-Time PCR experiments. "    Total Running Time:  9:52 (posted 10/29/2009)
    Direct YouTube link => http://www.youtube.com/watch?v=zm9QoIpOzkM
  • MIQE checklist
    http://www.helixis.com/support/usefultools/MIQE_Checklist.pdf
    Description: To help you follow the latest  MIQE guidelines, Helixis has formatted this useful checklist to keep handy at your bench or desk when designing your Real-Time PCR experiments or drafting your next paper.

  • New Standards for qPCR and RT-qPCR
    September 3, 2009 by Isobel
    Arguably, no technique has had greater impact on the progress of biomedical research in recent years than quantitative real-time PCR. It has accelerated the pace of research and opened up exciting possibilities for detection and treatment of disease. The widepread adoption of qPCR as a standard technique is evident even in the most cursory literature search; the term “real-time PCR” returns over 14,000 papers published in 2009 alone. However, many scientists are concerned about the lack of standardization of qPCR experiments.


    Quality assessment is a big fat elephant sitting in the room: everyone knows what needs to be done, but most researchers do not follow basic quality control guidelines. This serves to undermine the integrity of the scientific literature to such an extent, that a high proportion of publications are reporting technical or analytic artifacts”. Prof. Stephen Bustin, April 2009 SciTopics article.

The publication of a comprehensive set of guidelines for quantitative, real-time PCR highlights a need for greater consistency and standardization in reporting the results of qPCR and RT-qPCR analyses. The MIQE (Minimum Information for Quantitative Real-Time PCR) guidelines, published in the April 2009 edition of Clinical Chemistry, seek to create global consensus on how to best perform qPCR experiments and how to report qPCR results.

The paper, published by an international group of scientists from institutions throughout Europe and the United States, seeks to address issues from basic nomenclature (Cq, vs Ct, Cp, or TOP) to quality control of nucleic acids to oligo design and assay normalization. Some of the topics covered in detail are:

    • Sample processing
    • DNA and RNA quality and integrity
    • Appropriate controls
    • Comprehensive reporting of reagents, plasticware and protocols
    • Inclusion of oligo sequences and accession numbers of target genes
    • Publication of primer sequences
    • Considerations of target secondary structure and specificity of oligos for target
    • Inclusion of information on the validity of the reference genes for the sample type used
    • Comprehensive reporting of data analysis methods

The stated aim of the guidelines is to support the integrity of the scientific literature, promote consistency between labs, and increase experimental transparency. The MIQE checklist contains a list of mandatory and required elements that at first glance is somewhat daunting. However, the required elements are clearly there to help consistency, transparency, accuracy and reproducibility, and many of them, such as reporting the accession number of the target gene, and accurately identifying the reagents and protocols used, do not appear to be cumbersome. Most address the need for commonsense controls, accurate descriptions of sample handling processes, and consistency in nomenclature and normalization of results.

A related GEN article, published in August states: "Adoption of the mandatory guidelines as a first strategy assures that key parameters affecting data quality are being addressed immediately and will have a swift impact on confidence levels in the data and the conclusions drawn from it”

The authors are actively seeking feedback from the research community on these guidelines and consider them to be a constantly evolving document that is very much a work in progress.

  • Do Your RT-qPCRs Make The Grade?
    26th July 2009 - Real-time PCR is a technique that is now commonly employed in almost all molecular biology laboratories to quickly answer very specific questions. Northern and Southern blotting are now a thing of the past. No longer do we wait days to know whether a gene is expressed. We can have the answer in 45 minutes!
    But with the widespread use of such a wonderful and sensitive technology comes differences in how results are reported in the literature. There are also differences between reviewers reading these papers and their understanding of the essential information required to judge the accuracy of the reported data.
    To overcome this increasing problem of lack of consistency in publications, a panel of real-time PCR experts published a set of guidelines containing what they consider the minimal information required when reporting qPCR results. That paper called The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, was published February 2009 in the Journal of Clinical Chemistry.
    This is not only a great resource for authors, but it also essentially a troubleshooting guide as well. If you don’t have an answer to each of the item on the checklist, then maybe you are missing an essential piece of information in your experiment.
  • Publishing Data That Conform to the MIQE Guidelines
    Minimum information for publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines help researchers design qPCR experiments.
    Real-time quantitative polymerase chain reaction (qPCR) is a definitive technique for quantifying differences in gene expression levels between samples. However, a lack of consistency in experimental design and reporting combined with inadequate guidelines to review submitted articles with qPCR data greatly increases the potential of reporting statistically insignificant and conflicting results.1 The publication2 and retraction3 of a Science “Breakthrough of the Year 2005” article underlines the issue.

  • MIQE Guidelines 'Slowly Filtering Through' PCR Community Despite Lack of Journal Enforcement
    by Bernadette Toner Genome Web
    Guidelines proposed in early 2009 to help standardize how qPCR results are reported are "slowly filtering through" the research community, but much work still needs to be done to improve the quality of published qPCR studies, according to one of the authors of the standard.

  • Are your qPCR experiments compliant with MIQE?
    The MIQE guidelines establish specifications for the minimum information that must be reported for a qPCR experiment in order to ensure its relevance, accuracy, correct interpretation and repeatability.  Comply with MIQE guidelines !
    Learn why Prof. Kubista from the TATAA Biocenter uses the Agilent 2100 Bioanalyzer for RNA quality control  =>  Start webinar

  • Feature Article - PCR Technology Review:
    Standardization of qPCR and RT-qPCR - New Guidelines Seek to Promote Accurate Interpretation of Data and Reliable Results
    by Stephen A. Bustin,   Jo Vandesompele,   Michael W. Pfaffl
        =>   download PDF
    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” report. 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 conclusions.

  • A practical approach to RT-qPCR - Publishing data that conforms to the MIQE guidelines
    (Bio-Rad amplification tech note 5859)
    by Sean Taylor, et al., Bio-Rad Laboratories, Hercules, CA

  • MIQE Guidelines - RNA Qualitätskontrolle in der Genexpressionsanalytik – ein Meilenstein auf dem Weg zum Erfolg (in German) by Christiane Becker, Irmgard Riedmaier, and Michael W. Pfaffl
    Abstrakt (D) - Die Qualität des Probenmaterials, also der Gesamt-RNA, hat einen markanten Einfluss auf die Richtigkeit der quantitativen RT-PCR. Die Überprüfung der RNA Qualität vor einer Expressionsmessung ist unabdingbar, um verlässliche RT-qPCR Expressionsergebnisse zu erhalten.
    Abstract (E) - The integrity of total RNA has a distinct influence on the accuracy of RT-qPCR. Quality assessment is an essential step for the evaluation of reliable results in gene expression analysis.

  • Press release
    Standardization of qPCR and RT-qPCR - New Guidelines Seek to Promote Accurate Interpretation of Data and Reliable Results
    http://pressemitteilung.ws/node/166061

  • International Scientists Secure Quality in Molecular Diagnostics
    SALT LAKE CITY, March 31, 2009 - ARUP Laboratories and the American Association for Clinical Chemistry (AACC) announced today that a consensus guideline for a key laboratory method called qPCR (or quantitative polymerase chain reaction) was published by a group of international scientists representing the medical and research fields.
  • Consensus Guideline Reached For Quantitative Polymerase Chain Reaction
    Press release by TATAA Biocenter

    Gothenburg, March 31, 2009 - TATAA BIOCENTER and the American Association for Clinical Chemistry (AACC), announced today that a consensus guideline for a key laboratory method called qPCR (or quantitative polymerase chain reaction) was published by a group of international scientists representing the medical and research fields.
  • Internationale Wissenschaftler sorgen für Qualitätssicherung in der Molekulardiagnostik
    Salt Lake City (ots/PRNewswire) - - Einigung über Konsensus-Richtlinie bezüglich der quantitativen Polymerase-Kettenreaktion erzielt ARUP Laboratories und die American Association for Clinical Chemistry (AACC) gaben heute bekannt, dass eine Konsensus-Richtlinie für die wichtige, qPCR (quantitative Polymerase-Kettenreaktion) genannte Labormethode veröffentlicht worden sei. Verantwortlich für die Veröffentlichung war eine Gruppe internationaler Wissenschaftler als Vertreter der Gebiete Medizin und Forschung.
  • Real-timePCR data markup language
    The aim of MIQE, coordinated by a group of research-active scientists and coordinated under the umbrella of MIBBI (Minimum Information for Biological and Biomedical Investigations http://www.mibbi.org) is to provide authors, reviewers and editors specifications for the minimum information that must be reported for a qPCR experiment in order to ensure its relevance, accuracy, correct interpretation and repeatability. A checklist, which should be submitted along with the paper, is available for authors in preparing a manuscript employing qPCR.
    http://www.rdml.org/guidelines.php

  • Letter of the MIQE authors
    Letter to leading journals recommending the use of MIQE for quality control of qPCR experiments. 
    Download letter PDF

  • IBT of the Academy of Sciences of the Czech Republic
    PRAGUE, April 1, 2009 - Institute of Biotechnology of the Academy of Sciences of the Czech Republic, v.v.i. (IBT) and the American Association for Clinical Chemistry (AACC), announced today that a consensus guideline for a key laboratory method called qPCR (or quantitative polymerase chain reaction) was published by a group of international scientists representing the medical and research fields.
  • qPCR Grows Up by genome web
    Bustin is now at the forefront of a movement to get researchers to follow a set of guidelines, the minimum information for publication of quantitative real-time PCR experiments, or MIQE, that were published online at Clinical Chemistry in February.
    "In my talks, I always refer to the cowboy stage of qPCR. For quite a while everything went," Bustin says. In particular, he casts a critical eye on how people have been normalizing their gene expression data. In northern blot and standard PCR experiments that didn't give quantitative data, people often used a single reference gene. "People just moved that approach to qPCR without thinking about what they were doing," Bustin says. "Are these reference genes really invariant or are they changing with treatment?"
  • qPCR Assay Quality assessment on SciTopics
    8 April 2009 by Prof Stephen Bustin;  Category: Biochemistry, Genetics and Molecular Biology
    Guidelines for minimum information required for publication of qPCR data have now been published in Clinical Chemistry
    qPCR quality assessment relates mainly to the reverse transcription -qPCR (RT-qPCR) variant of the technology. This is widely used to measure pathogen as well as cellular RNA copy numbers; the former, given appropriate standard operating procedures and technical expertise, is fairly straightforward. The latter can be highly problematic. For both types of assay, however, RNA quality is a major consideraton.
    Quality assessment is a big fat elephant sitting in the room: everyone knows what needs to be done, but most researchers do not follow basic quality control guidelines. This serves to undermine the integrity of the scientific literature to such an extent, that a high proportion of publications are reporting technical or analytic artifacts.
    Incredibly, many researchers are not bothered by this; indeed some have been heard to remark that they can't be bothered assessing RNA quality, worrying about reverse transcription or determining what normalisdation strategy to follow. However, efforts are underway to establish a checklist for journal editors and reviewers, with the aim of introducing a minumum standard of assay reporting.
  • Quest Agrees to Pay Fine for Misbranding Tests
    First-ever consensus guidelines on quantitative PCR aim to improve the quality and transparency of studies involving qPCR (Clin Chem, 2009; 55: 611-622). The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines outline the minimum information necessary to evaluate qPCR studies, including all relevant experimental conditions and assay characteristics, and full disclosure of all reagents, sequences, and analysis methods. The guidelines include an 85-item checklist of desirable and essential steps to be followed when using qPCR and information to be divulged from experiments involving qPCR. The purpose of the guideline is to encourage better experimental practice, so as to enable more reliable and unequivocal interpretation of qPCR results.
    Use of qPCR has proliferated, yet studies “invariably use diverse reagents, protocols, analysis methods, and reporting methods,” the authors wrote. “This remarkable lack of consensus on how best to perform qPCR experiments has the adverse consequence of perpetuating a string of serious shortcomings that encumber its status as an independent yardstick.” If researchers follow the guidelines, they should be able to design and report qPCR experiments with greater inherent value, and fellow researchers, editors, and laboratorians should be able to evaluate the technical quality of the published data against an established standard.
  • Advancing DNA research safely and securely
    27 May 2009; Dr Jeremy Garson& Dr Jim Huggett - Dr Jeremy Garson (UCL Centre for Virology) and Dr Jim Huggett (UCL Centre for Infectious Diseases and International Health) have been at the heart of developing a new set of guidelines on the way scientists the world over use qPCR – a technology crucial to forensic analysis and diagnosing diseases. Below Dr Huggett explains how and why they went about it.
    What do you hope to achieve with the guidelines?
    By developing the MIQE guidelines, we aim to enable researchers to perform high-quality qPCR that allows their experiments to be easily understood and repeated by workers in laboratories anywhere in the world. For science to advance swiftly and securely it is essential that the results of experiments can be independently reproduced.
  • Data that Meets the MIQE Guidelines
    Canadian BioTechnologist 2.0 on 27 May 2009 - Key Steps to Generating High Quality Real-Time PCR (RT-qPCR) Data that Meets the MIQE Guidelines Speaker: Sean Taylor, Ph.D., Bio-Rad Laboratories PDF slide deck.
  • GLOSSARY OF REAL-TIME PCR TERMS   by   M.Tevfik Dorak
    MIQE - An initiative by the International Real-time PCR Data Markup Language (RDML) Consortium to generate a structured and universal data standard for exchanging quantitative real-time PCR experiment data. This effort resulted in standard guidelines for reporting qPCR data  (publication checklist:  XLSPDF)

2008
RDML guidelines (formaly known as MIqPCR)
Working draft, 4th April, 2008 - RDML guidelines (formaly known as MIqPCR)

It is crucial that data acquisition, analysis and reporting become more transparent to allow reinterpretation and to guarantee compliance with quality standards. Therefore, following the example of the microarray community and their MIAME (Minimum Information About a Microarray Experiment) guidelines, we propose guidelines specifying the minimal information about qPCR experiments. A RDML guidelines compliant RDML file should contain all measured data as well as information about the samples and targets being analyzed.

In addition, data must be linked to samples and targets in an unequivocal way. Due to the complexity and diversity of experiments in which qPCR is utilized, the scope of the RDML guidelines is limited to the technology itself, which means that these guidelines can easily be integrated into other minimum information guidelines that focus on the wider experimental context. To coordinate this effort, the RDML consortium recently joined the MIBBI project (Minimum Information for Biological and Biomedical Investigations). The minimum information guidelines have been kept minimal to facilitate the creation of a compliant RDML files that make the least demand on researchers’ time, while requiring sufficient information for other researchers to interpret and reanalyze the data contained within an RDML guidelines compliant RDML file.


2006
  • How Reliable is Your qPCR Data?
    Drug Discovery & Development - March 01, 2006

    These excerpts from a recent Webcast on quantitative polymerase chain reaction for gene expression analysis involve experts from industry and academia discussing their experiences with, and data gained from, the method.
    Senior Editor Patrick McGee recently hosted a Webcast entitled "How reliable is your qPCR data?" Quantitative PCR is a powerful and sensitive technology for the quantification and validation of genetic data. Despite the power of qPCR, however, a number of key considerations need to be addressed, from sample preparation through data analysis. Although the topic of the day was overcoming the challenges of qPCR, from pre-assay through data analysis, panelists limited their comments to gene expression analysis. The full version of the Webcast is available for viewing at www.dddmag.com/qpcr.
    The panel of experts who joined McGee for the Webcast included Stephen Bustin, PhD, professor of Molecular Science at Barts and the London Queen Mary's School of Medicine and Dentistry at the University of London. His research group focuses on molecular oncology and has spent the last eight years working on applying molecular techniques such as qRT-PCR to the biology of colorectal cancer. Mark Anderson, PhD, research and development scientist, Invitrogen Corp., has extensive experience in analyzing and developing PCR technology and he discussed qPCR assay design and troubleshooting. Maurice Exner, PhD, research and development manager in infectious diseases at Quest Diagnostics, is responsible for directing research efforts to develop new clinical diagnostic assays for infectious diseases. These assays primarily use automated nucleic acid extraction methods coupled with various nucleic acid amplification techniques, particularly qPCR.


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