MIQE media & press review: 2018 -- NEWS 2017 2016 2015 2014 2013 2012 2011 2010 2009 2008 2006		follow us on twitter    MIQE_qPCR    follow us on facebook   MIQE_qPCR MIQE and qPCR Qualiy Control talks on www.eConferences.de - Amplify your knowledge!
The MIQE Guidelines -- Minimum Information for Publication of Quantitative Real-Time PCR Experiments Introduction The MIQE key publications: The MIQE Guidelines - Minimum Information for Publication of Quantitative Real-Time PCR Experiments Why the need for qPCR publication guidelines?  -  The case for MIQE MIQE precis:  Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments Guidelines for Minimum Information for Publication of Quantitative Digital PCR Experiments How to do successful gene expression analysis using real-time PCR MIQE & qPCR iBook -- How to apply the MIQE guidelines - a visual, interactive and practical qPCR guide Improving biological relevancy of transcriptional biomarkers experiments by applying the MIQE guidelines to pre-clinical and clinical trials New MIQE papers by the MIQE research community RDML guidelines (formaly known as MIqPCR) qPCR Assay Quality assessment
		MIQE qPCR & dPCR How to apply the MIQE guidelines - a visual, interactive and practical qPCR & dPCR guide 5th Edition (20th February 2022) Editors:  Afif M. Abdel Nour & Michael W. Pfaffl ISBN 9783000488061 Free download via Apple Books (iBooks version) -- https://books.apple.com/us/book/id1610947651 Free download via GQ (ePub version) -- ePub (158 MB) Free download via GQ (PDF version) -- PDF (52 MB) Please let us know, if you want to participate with an own chapter in the next 6th edition of the MIQE qPCR & dPCR iBook, contact us via  iBook@bioMCC.com New book chapters in 5th eds:
		Chapter 14 Development of a high-throughput data analysis method for evaluating quantitative real-time PCR (qPCR) assays by Gregory C. Patton, Ph.D., Andrew N. Gray, Ph.D., Nathan A. Tanner, Ph.D., Janine G. Borgaro, Ph.D., Yan Xu, Ph.D., Julie F. Menin, M.S. & Nicole M. Nichols, Ph.D., New England Biolabs, Inc. Quantitative real-time PCR (qPCR), first demonstrated by Higuchi and colleagues (Higuchi et al., 1992), is an essential molecular biology technique for detecting and quantifying nucleic acids and a mainstay of molecular diagnostics workflows. Workflow simplicity and advances in instrumentation now permit sizeable quantities of data to be generated rapidly, with 96, 384, or even 1536 reactions in a single qPCR experiment. These experiments require thoughtful design and analysis to capture all relevant information, such that accurate and appropriate conclusions can be drawn. We sought to develop a suite of general use qPCR reagents that maintained performance across various targets and workflow parameters to simplify downstream assay design complexity. This development project involved repeated data collection on a series of test panels, each containing multiple targets. Target were chosen to represent parameters that have been shown to impact assay performance, including variations in primers (e.g., length, GC content, location) and targets (e.g., length, GC content, transcript abundance). Consistent with MIQE guidelines, standard curves were used to evaluate test panel assay performance, but after reviewing efficiency, reproducibility and non-template controls from numerous experiments, it became clear that a more scalable approach to data analysis and visualization was required to better understand how changes in reagent composition impacted performance. To compare various amplicon panels over multiple qPCR runs, instruments, reagents and conditions, a high-throughput data analysis method termed “dots in boxes” was developed. The output of this analysis captures key assay characteristics, including those highlighted in MIQE guidelines, as a single data point for each qPCR target. This method of analysis permits multiple targets and conditions to be compared in a single graph, allowing concise visualization and rapid evaluation of overall experimental success.
		Chapter 20 Digital Microfluidic PCR in QIAcuity Nanoplates Kevin Matthaei, Christina Goebel, Michael Bussmann, Gerald Schock, Afif Abdel Nour, Andreas Missel; QIAGEN GmbH QIAcuity systems are designed to determine absolute amounts of target DNA in a sample using a digital PCR (dPCR) approach. Digital PCR uses the procedure of end-point PCR but splits the PCR reaction into many single partitions in which the template is randomly distributed across all available partitions. After PCR, the target molecule is detected by measuring the fluorescence – of either sequence-specific DNA probes or intercalating dyes – in all positive partitions. As the template is distributed randomly, Poisson statistics can be used to calculate the amount of target DNA per positive partition. The total amount of target DNA in all partitions of a well is then calculated by multiplying the amount of target DNA per partition with the number of positive partitions. Measurement of target concentration is determined based on the volume in all analysable partitions, i.e., partitions filled with reaction mix, as identified by a fluorescent dye present in the reaction mix itself. Absolute quantification by dPCR eliminates the need for standard curves to determine amounts of target DNA in a given sample. Aside from absolute quantification, the QIAcuity Software Suite provides analysis modules for mutation detection, genome editing analysis, copy number variation (CNV), and gene expression analysis.
		MIQE & qPCR iBook How to apply the MIQE guidelines - a visual, interactive and practical qPCR guide 4th Edition (8th June 2020) Editors:  Afif M. Abdel Nour & Michael W. Pfaffl ISBN 9783000488061 Free download via iTunes or Apple Books -- https://books.apple.com/de/book/MIQE-qPCR/id993276375 Download the interactive PDF -- PDF (47 MB) Please let us know, if you want to participate with an own application or chapter in the MIQE & qPCR iBook, contact us via  iBook@bioMCC.com _______________________ Editorial for 4th edition June 2020 The success story of the qPCR and dPCR MIQE guidelines is continuing! Editorial for 4th edition published in June 2020 by Afif M. Abdel Nour & Michael W. Pfaffl Yes the story is continuing! More and more researchers, biological journals, academic and commercial institutions worldwide, are supporting the MIQE guidelines. The very high recitation record of Scopus and Google Scholar documents this. As for June 2020, we count multiple thousand citations for the qPCR MIQE guideline (7.350 by Scopus and 9.800 by Google Scholar) as well as for the digital MIQE guideline (370 by Scopus and 460 by Google Scholar). Hence, the qPCR and dPCR MIQE guidelines are a worldwide standardisation success story, which are driven forward by scientific validity and credibility in the PCR community. The present 4th edition of the MIQE & qPCR iBook will push the ‘MIQE idea’ even further in any laboratory worldwide and beyond in the scientists’ workflow and minds. It should clearly show how to apply the guidelines and serve as a handy, visual, interactive and practical guide. Since the 2nd edition in July 2016, we provided an additional MIQE & qPCR eBook version, which is readable on any eBook reader device (ePub file). This leaded to more than 4.800 extra downloads of the MIQE & qPCR iBook and eBook from over 40 countries worldwide. Our goal for the 4th edition is to update the existing content by new hot topics, and to create an overall fancy interactive tool, by interfacing scientific publications with educating pictures, videos and scientific talks. The editors implemented the following new chapters: “ddPCR – droplet digital PCR” authored by Afif Abdel Nour "Reproducible and Sensitive Assays using 3- and 6-colour Crystal Digital PCR™ for Detecting Point Mutations in Human Breast Cancer" authored by scientists from Stilla Technologie "The influence of plastic consumables on qPCR" authored by application specialists by Eppendorf In summary, we are proud to present a selection of international highly recognized authors from the academic field and from industrial research, presenting their latest research applications. The described qPCR and dPCR methods and applications are tightly linked to the MIQE context, and show it clearly based on educational questionnaires or interactive ‘how to do’ instruction sheets. The at-hand MIQE & qPCR iBook & eBook should deliver the MIQE guidelines directly to the researcher and help to solve the daily problems in the molecular biology laboratory using quantitative PCR, digital PCR, single-cell qPCR, microRNA applications or any comparable techniques using PCR. The editors hope you like our explanatory, interactive and educational iBook, eBook and ePub concept, showing the advantages of the MIQE guidelines in an easy and understandable way. The proper application and recommendation mentioned in that publication should guarantee the successful qPCR or dPCR application in the lab, and will help authors, reviewers, editors, and researchers to evaluate the quality of the presented publication. The editors Afif M. Abdel Nour & Michael W. Pfaffl
MIQE Guidelines -- Chinese translation MIQE Guidelines -- Japanese translation MIQE Guidelines -- Arabian translation

2018

• More than 7.000 citations of the MIQE paper!
  https://scholar.google.de/scholar?oi=bibs&hl=de&cites=6338124712618390161
• MIQE guidelines  -- Technical aspects and recommendations for single-cell qPCR
  
  Quantitative PCR is a rather mature technology and MIQE (Minimum Information for publication of Quantitative real-time PCR Experiments) guidelines are available for its use on classical bulk samples (Bustin et al., 2009). Similar guidelines are also provided for digital PCR (Huggett et al., 2013). The goal of MIQE is to provide the basis for good experimental practice and encourage detailing protocols that allow for unbiased interpretation of qPCR data. Most aspects of the MIQE guidelines are relevant for single-cell analysis, but some experimental steps need adjustments and there are also some unique steps in single-cell analysis that must be considered. Table 2 shows the additional and adjusted items that are specific to single-cell analysis.
  
• Nice MIQE webpage from Sigma-Aldrich (now a Merck company)
  MIQE – Minimum Information for Publication of Quantitative Real-Time PCR Experiments
  ... and about PCR and qPCR:
  
  • Introduction and Historical Timelines
  • Polymerase Chain Reaction
  • Quantitative PCR
  • Digital PCR
  • Quantitative PCR and Digital PCR Detection Methods
  • PCR qPCR dPCR Assay Design
  • Sample Purification and Quality Assessment
  • Reverse Transcription
  • Assay Optimization and Validation
  • Data Analysis
  • Troubleshooting
  • Regulatory Agencies and Conclusions
  • Additional Resources
  • Protocols

2017

• Free access to the MIQE talks held at the 8th Gene Quantification Event in April 2017 
  

• NEW in October 2017 --  Download ePub Version for any eBook reader
  MIQE & qPCR iBook -- How to apply the MIQE guidelines - a visual, interactive and practical qPCR guide
  2nd Edition (version 2.5 published 12th December 2016)
  Editors:  Afif M. Abdel Nour & Michael W. Pfaffl
  ISBN 9783000488061
  Free download of the iBook via iTunes -- https://itunes.apple.com/book/miqe-qpcr/id993276375?mt=11
• Positioning Digital PCR for Sharper Genomic Views
  To Get Even More Out of Weak Signals, dPCR Is Deploying Precision Fluidics, Selective Primers, and Powerful Analytics
  In the past decades, a diverse set of protocols, instruments, and analysis methods generated volumes of qPCR data—data not easily amenable to meta-comparisons. The need for consensus on best practices led to establishment of optimal experimental guidelines, or Minimum Information for Publication of Quantitative Real PCR Experiments (MIQE). The guidelines defined several performance metrics to ensure assay robustness and reproducibility, such as PCR efficiency, dynamic range, limit of detection, target specificity, and precision.
  
• Primerdesign qPCR Tips | Follow the MIQE Guidelines
  Welcome to Primerdesign qPCR Tips: In 2009, After major scientific scandals caused by poor qPCR data, key opinion leaders in the field defined the critical requirements for a good qPCR experiment in a seminal paper. We recommend the MIQE guidelines.  As the definitive guide to producing publication quality qPCR data. Even if you cant implement every recommendation, it will put your work on a firm footing with other scientists and reviewers.
  
• Talking the talk, but not walking the walk --   RT-qPCR as a paradigm for the lack of reproducibility in molecular research
  Stephen Bustin and Tania Nolan
  Eur J Clin Invest. 2017 47(10): 756-774
  
  Poorly executed and inadequately reported molecular measurement methods are amongst the causes underlying the lack of reproducibility of much biomedical research. Although several high impact factor journals have acknowledged their past failure to scrutinise adequately the technical soundness of manuscripts, there is a perplexing reluctance to implement basic corrective measures. The reverse transcription real-time quantitative PCR (RT-qPCR) is probably the most straightforward measurement technique available for RNA quantification and is widely used in research, diagnostic, forensic and biotechnology applications. Despite the impact of the minimum information for the publication of quantitative PCR experiments (MIQE) guidelines, which aim to improve the robustness and the transparency of reporting of RT-qPCR data, we demonstrate that elementary protocol errors, inappropriate data analysis and inadequate reporting continue to be rife and conclude that the majority of published RT-qPCR data are likely to represent technical noise.
• QUANTITATIVE PCR RESULTS REMAIN QUESTIONABLE -- THE NEED FOR THE MIQE GUIDELINES
  Basic Science Update -- Technology
  May 13th 2017 -- Talk by Prof. Stephen A. Bustin
  
  

• Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets -- from variable nonsense to publication quality data
  Sean C. Taylor, Genevieve Laperriere, Hugo Germain
  Scientific Reports 7, Article number: 2409 (2017)
  
  Quantitative PCR (qPCR) has become the gold standard technique to measure cDNA and gDNA levels but the resulting data can be highly variable, artifactual and non-reproducible without appropriate verification and validation of both samples and primers. The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing. The most susceptible, frustrating and often most interesting samples are those containing low abundant targets with small expression differences of 2-fold or lower. Here, Droplet Digital PCR (ddPCR) and qPCR platforms were directly compared for gene expression analysis using low amounts of purified, synthetic DNA in well characterized samples under identical reaction conditions. We conclude that for sample/target combinations with low levels of nucleic acids (Cq ≥ 29) and/or variable amounts of chemical and protein contaminants, ddPCR technology will produce more precise, reproducible and statistically significant results required for publication quality data. A stepwise methodology is also described to choose between these complimentary technologies to obtain the best results for any experiment.
  
• MIQE: A Step Toward More Robust and Reproducible Quantitative PCR
  Stephen A. Bustin and Carl T. Wittwer
  Clinical Chemistry 2017 63(7) Published July 2017
  Featured Article -- The MIQE guidelines: minimum information for publication of quantitative realtime PCR experiments.
  Bustin SA et al. Clinical Chemistry 2009;55:611–22.3
  
  The concept of using in vitro enzymatic synthesis to amplify DNA was first mooted in 1971 (1) and demonstrated in 1985 as the “polymerase chain reaction” (2). PCR enables the detection of a unique DNA sequence amongst a vast background of other, similar DNA molecules. Its remarkable combination of conceptual simplicity and practical accessibility, together with the addition of reverse transcription for detection of RNA, and continuous improvements to reagents, protocols, and instruments has secured PCR’s status as today’s most versatile and ubiquitous molecular laboratory technique. In its original guise as an end-point assay, “endpoint PCR” required gel electrophoretic analysis. This method was time-consuming, limited in analytical sensitivity, dynamic range, and resolution, and introduced the potential for contamination. Importantly, endpoint PCR was nonquantitative. The introduction of real-time fluorescencebased quantitative PCR (qPCR) changed this by detecting PCR amplicons during the exponential phase using fluorescent reporters (3). With this feature, coupled with no processing after PCR, less contamination, and statistical analyses, qPCR became the method of choice for quantitative applications.
  
  However, such popularity created a myriad of different protocols, reagents, and analysis methods, which, when combined with different nucleic acid extraction and quality assessment methods, resulted in the publication of implausible and contradictory results. Importantly, the omission of detailed technical information made it challenging to gauge the soundness of qPCRbased results. Specifically, nucleic acid integrity and purity assessments were rarely reported, variability introduced by the reverse transcription step was disregarded, PCR efficiencies were not specified, and normalization procedures were not justified. Publication of erroneous conclusions in the scientific literature became commonplace.
  
  One egregious example of qPCR misuse ostensibly supported a link between measles virus and gut pathology in autistic children. A detailed examination of the qPCR evidence revealed poor assay design, widespread disregard of control results suggesting contamination issues, inadequate nontransparent reporting, and questionable data analysis (4). This provided the final impetus for a group of international scientists to introduce recommendations for qPCR assay design and data reporting. A best practice, commonsense approach of minimum guidelines in categories critical for obtaining reliable results was published in the article discussed here under the acronym MIQE,which stands for “minimum information for the publication of qPCR experiments.” This publication is now the second most cited paper in Clinical Chemistry.
  
  The MIQE guidelines are now the accepted standard for both optimal qPCR assay design and transparent reporting, actively championed by PCR reagent and instrument manufacturers and many journals. They have also been adapted for diagnostic and clinical applications (5). Furthermore, the increasing popularity of digital PCR has resulted in the publication of MIQE guidelines for digital PCR (6) and the challenges of RNA sequencing have resulted in MIQE-inspired guidelines (7).
  
  However, the majority of qPCR publications still do not provide sufficient technical detail (8) and, where information is provided, it often invalidates the authors’ conclusions (9). Many high–impact factor journals appear to have improved technical reporting standards, but there is actually less transparency today (10). Clearly, technical guidelines are an important step, but unless the scientific community as a whole takes more responsibility, it will continue to be plagued with immaterial, misleading, time- and money-wasting conclusions. The question remains: if a technique as “simple” as qPCR is handicapped by inappropriate use and inadequate scrutiny, what hope is there for the more complex technologies in use today?
• Following the MIQE Guidelines for RT-qPCR Experiments
  from Bio-Rad Life Sciences via Labtube TV
  For more info, visit  http://www.bio-rad.com/yt/1/MIQE-for-RT-qPCR
  
  Researchers – and journals – have been slow to adopt the MIQE guidelines that were established in 2009 to bolster the reliability of real-time PCR (qPCR) and reverse transcription qPCR (RT-qPCR) data. In response, Sean Taylor and Eli Mrkusich wrote a brief and practical guide that concisely summarizes the key steps required to produce high quality, reproducible data for labs conducting RT-qPCR experiments. This video highlights the key steps required to produce reliable and reproducible qPCR data as described in the article, published in the Journal of Molecular Microbiology and Biotechnology in November, 2013. Bio-Rad's PrimePCR™ 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). These DNA primer pairs were designed by prioritizing the gene regions most commonly found in transcript variants. Strict design criteria were used to ensure optimal real-time PCR results for each target: • Target regions without SNPs • PCR primer pairs annealing across intron/exon junctions when possible • No secondary structure in primer annealing sites • Maximum number of transcript isoforms detected • PCR primers compatible with standard assay conditions.
  
• Are MIQE Guidelines Being Adhered to in qPCR Investigations in Photobiomodulation Experiments?
  Nicolette N. Houreld, DTech
  Photomedicine and Laser Surgery 2017 35(2): 69-70
  The polymerase chain reaction (PCR) is an important and reliable technology for research and diagnostic analysis, and is a quick and easy method of enzymatically synthesizing and amplifying unlimited copies of specific DNA sequences in a few short hours. From its inception, PCR has matured over the years from a laborious, timeconsuming, and gel-based technique to an automated, high throughput, rapid quantitative technique. This technique, which formed the cornerstone of the human genome project, was only developed 20 years ago. The technique as we know it originates from research conducted in the 1980s at Cetus Corporation in California.
• Reproducibility of biomedical research - The importance of editorial vigilance.
  Bustin SA and Huggett JF
  Biomoleculoar Detection Quantification 2017 (11): 1-3
  Many journal editors are a failing to implement their own authors' instructions, resulting in the publication of many articles that do not meet basic standards of transparency, employ unsuitable data analysis methods and report overly optimistic conclusions. This problem is particularly acute where quantitative measurements are made and results in the publication of papers that lack scientific rigor and contributes to the concerns with regard to the reproducibility of biomedical research. This hampers research areas such as biomarker identification, as reproducing all but the most striking changes is challenging and translation to patient care rare.
  

2016

• The qPCR and dPCR MIQE guidelines – A success story!
  

  The MIQE guidelines and the resulting scientific validity will be supported by more and more researchers, biological journals, academic and commercial institutions. Today late November 2016 we count 5000 citations for the MIQE guideline applied in qPCR and 185 citations for the digital PCR (dPCR) MIQE guideline (measured by Google Scholar). Hence the qPCR and dPCR MIQE guidelines are a worldwide full success story which will be driven forward by the scientific community.
• The importance of correctly controlled qPCR
  
  Now Available On Demand -- Despite substantial advances in the accessibility and ease-of-use of PCR for diagnostics, generating an assay that delivers reliable, reproducible and meaningful results is still a challenging task.
  
  We will explore:
  How the Eco 48 real-time quantitative PCR (qPCR) Thermal Cycler from PCRmax can be used for rapid development of robust bacterial and fungal assays.
  How to rely on the speed and precision of this technology for a range of assay applications, from quality assurance through to Intraopperative PCR.
  "Minimum Information for Publication of Quantitative real-time PCR Experiments" (MIQE) guidelines from author Stephen Bustin, Professor of Molecular Medicine, Anglia Ruskin University.
  
• The focus on sample quality: Influence of colon tissue collection on reliability of qPCR data.
  Korenkova V, Slyskova J, Novosadova V, Pizzamiglio S, Langerova L, Bjorkman J, Vycital O, Liska V, Levy M, Veskrna K, Vodicka P, Vodickova L, Kubista M, Verderio P
  Sci Rep. 2016 6: 29023
  
  Successful molecular analyses of human solid tissues require intact biological material with well-preserved nucleic acids, proteins, and other cell structures. Pre-analytical handling, comprising of the collection of material at the operating theatre, is among the first critical steps that influence sample quality. The aim of this study was to compare the experimental outcomes obtained from samples collected and stored by the conventional means of snap freezing and by PAXgene Tissue System (Qiagen). These approaches were evaluated by measuring rRNA and mRNA integrity of the samples (RNA Quality Indicator and Differential Amplification Method) and by gene expression profiling. The collection procedures of the biological material were implemented in two hospitals during colon cancer surgery in order to identify the impact of the collection method on the experimental outcome. Our study shows that the pre-analytical sample handling has a significant effect on the quality of RNA and on the variability of qPCR data. PAXgene collection mode proved to be more easily implemented in the operating room and moreover the quality of RNA obtained from human colon tissues by this method is superior to the one obtained by snap freezing.
• Video tutorials MIQE Guidelines
  "MIQE Oldies but Goodies" by Sigma Aldrich and co-workers
  
  • MIQE -- Concepts
  • MIQE -- RNA Quality Considerations
  • MIQE -- RNA Quantity and RT Considerations
  • MIQE -- Assay Design Considerations
  • MIQE -- Sample Derived Inhibitors
  • MIQE -- Reference Gene Validation
  • MIQE -- Data Analysis Guidelines
  • MIQE -- Assay Design Considerations
  • MIQE -- Sample Derived Inhibitors
• Quality control for the quantification of gene expression biomarkers.
  by Jens BJÖRKMAN, TATAA Biocenter
  Quality matters: Improving the quality of biological resources
  Session 3: Quality control in Genomics @ Biobanking National Infrastructure Meeting
  WEDNESDAY MAY 18, 2016
  
  Measuring an RT-qPCR signal with an assay targeting a transcript of interest is easy and the data generated often look good, but it is a different story if the signal truly reflects the amount of targeted transcript that actually were present in the sample in vivo. This has to be validated to greatest extent possible by performing relevant controls. I will describe quality control measures to test for degradation of RNA, inhibition, genomic DNA background and also provide means to compensate for interplate variation.
  
• Digital polymerase chain reaction for characterisation of DNA reference materials
  Somanath Bhat, , Kerry R. Emslie
  Biomolecular Detection and Quantification; Available online 3 May 2016
  
  Accurate, reliable and reproducible quantification of nucleic acids (DNA/RNA) is important for many diagnostic applications and in routine laboratory testing, for example, for pathogen detection and detection of genetically modified organisms in food. To ensure reliable nucleic acid measurement, reference materials (RM) that are accurately characterised for quantity of target nucleic acid sequences (in copy number or copy number concentration) with a known measurement uncertainty are needed. Recently developed digital polymerase chain reaction (dPCR) technology allows absolute and accurate quantification of nucleic acid target sequences without need for a reference standard. Due to these properties, this technique has the potential to not only improve routine quantitative nucleic acid analysis, but also to be used as a reference method for certification of nucleic acid RM. The article focuses on the use and application of both dPCR and RMs for accurate quantification.
• New digital PCR reporting guidelines for molecular diagnostics
  National Measurement System
  LGC scientists have collaborated on the development of best practice guidelines for the reporting of digital PCR data. Digital PCR is an emerging tool for DNA analysis showing great promise in new challenging areas of clinical diagnostics. These reporting guidelines provide a gold standard checklist of experimental information that should be included in all digital PCR publications to enable the research community to review and compare.

• Top 5 reasons your qPCR data was rejected!
  An insider perspective by Roche Life Science
  
  You finally finished all the replicate experiments, analyzed and reanalyzed the data, wrote and revised the manuscript, and completed the dreaded online submission steps. And you wait. And wait. Then, after weeks of anxiously (or compulsively) checking your email for communication from the editorial office, you finally receive official word that your paper was rejected. After a few tears, interspersed by stuttered whimpers, and a few textbooks thrown against the wall, you pull yourself together and plan your next move. After all, what kind of scientist would you be without facing adversity in the all-too-familiar form of rejection?
  
  Unfortunately, this is not an uncommon phenomenon in the realm of basic science manuscripts, and in particular, with qPCR-based studies. However, there are strategies to improve your chances of success and maximizing presentation of your qPCR data. Therefore, in this article, we will discuss the top five reasons your qPCR data was rejected and how to combat these pitfalls:
  
    1. Insufficient quantitation
    2. Incomplete methods
    3. Lack of MIQE compliance
    4. Scientific writing
    5. Biological relevance
  
• MIQE compliance in expression profiling and clinical biomarker discovery
  Irmgard Riedmaier, Melanie Spornraft, Benedikt Kirchner and Michael W. Pfaffl
  Technical University of Munich
  European Pharmaceutical Review VOLUME 20 (6)
  
  Molecular diagnostics and biomarker discovery are gaining increasing attraction in clinical research. This includes all fields of diagnostics, such as risk assessment, disease prognosis, treatment prediction and drug application success control1,2. The detection of molecular clinical biomarkers is very widespread and can be developed on various molecular levels, like the genome, the epi-genome, the transcriptome, the proteome or the metabolome. Today, numerous high-throughput laboratory methods allow rapid and holistic screening for such marker candidates. Regardless of which molecular level is analysed, in order to detect biomarker candidates, high sample quality and a standardised and highly reproducible quantification workflow are prerequisites. This article describes an optimal and approved development strategy to discover and validate ‘transcriptional biomarkers’ in clinical diagnostics, which are in compliance with the recently developed MIQE guidelines3. We focus on the importance of sample quality, RNA integrity, available screening and quantification methods, and biostatistical tools for data interpretation.
• Pitfalls in PCR troubleshooting: Expect the unexpected?
  Schrick L and Nitsche A
  Biomol Detect Quantif. 2016 Jan; 6: 1–3  (eCollection 2016)
  PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings.
  

A summary of MIQE relevant talks presented by eConferences.de

• Improve the Quality of your qPCR Result -- Learn how RNA normalization using PIPETMAX® easily improves the quality of qPCR results
  March 2016 by Gilson
  Normalization of RNA concentrations prior to reverse transcription reduces error in qPCR experiments, and is recommended by the MIQE guidelines for qPCR experiments.
  
  PIPETMAX® Normalization Assistant provides a flexible interface that everyone in the lab can use to create fully automated DNA or RNA normalization protocols, effortlessly incorporating normalization into your workflow while maintaining the integrity of your important nucleic acid samples. Adding PIPETMAX® qPCR Assistant further improves the traceability and reliability of your qPCR sample prep by fully automating master mix prep, sample dilutions, and qPCR plate preparation – all at your fingertips on PIPETMAX®.
  
  When integrated into your workflow, these two automated lab assistants provide reliable pipetting and verifiable results while minimising bookkeeping errors and user variation, saving precious time and delivering the meaningful qPCR results your research demands.
  Want to learn more about using MIQE guidelines and automation for more reproducible, meaningful qPCR data?
  Download the MIQE & qPCR iBook – How to Apply the MIQE Guidelines - A Visual, Interactive and Practical qPCR Guide. This free iBook is available for anyone who is interested in implementing automation and MIQE guidelines to qPCR workflows to improve the reproducibility and reliability of his or her results.
  Covering a range of issues associated with this powerful and sensitive molecular biology technique, the guide addresses the increasing need for traceability and compliance with MIQE guidelines in both regulated and non-regulated lab environments.Advanced automation solutions are discussed with respect to the advantages they bring to molecular diagnostic and basic research laboratories, including improved sample traceability, precise pipetting performance, and both qualitative and quantitative control measures.
  Topics addressed include:
  
  • Factors that affect verifiability of qPCR results
  • Advantages of implementing standardised, quality controlled qPCR methods and practices
  • Improvement in sample traceability and data reproducibility with automated workflows
  • Shortcomings of current software solutions.
• Improving Real-Time PCR Data Quality -- Developing an Assay to Deliver Reliable and Reproducible Results with Novel Instrumentation
  Drug Discovery Tutorial
  Feb 1, 2016 (Vol. 36, No. 3) by Andrew Birnie
  
  The polymerase chain reaction (PCR) has radically transformed biological science, allowing sophisticated analysis of genes and the genome. Revolutionizing the study of DNA, PCR is often hailed as one of the most important scientific advances of the 20th century. Over time, PCR has evolved into fluorescence-based quantitative real-time PCR (qPCR), which is now considered the molecular diagnostic technique of choice due to its capacity to detect and measure minute amounts of nucleic acids in a variety of samples from multiple sources.
  
  Due to its practical simplicity, in combination with its outstanding capabilities, including speed, sensitivity, and specificity, qPCR plays a huge role in a number of applications, among them gene expression analysis, microRNA analysis, single nucleotide polymorphism genotyping, copy number variation analysis, and protein analysis.The success and reputation of qPCR is reflected in the abundance of publications reporting qPCR data. Despite substantial advances in the accessibility and ease-of-use of qPCR for diagnostics, generating an assay that is capable of delivering reliable, reproducible, and meaningful results is still a challenging task.
• PCR and the MIQE Guidelines
  Presentation on PREZI
  
• Research focused on the polymerase chain reaction - comparative analysis and end user preference study
  Published 30 March 2016 by Rohan Salgarkar
  
  The overall market for PCR technology market is expected to grow rapidly owing to the expansion of its application areas and increased accuracy and precision. Furthermore, specific technology segments of the PCR market, such as dPCR (real-time PCR) and qPCR (digital PCR), are witnessing higher market growth owing to their benefits such as real-time process monitoring, low reagent consumption, automation of workflow, and greater reproducibility and precision.
  

2015

• Perspective -- Improving the reliability of peer-reviewed publications: We are all in it together.
  by Stephen A. Bustina & Tania Nolana
  Biomolecular Detection and Quantification (available online 28 December 2015)
  The current, and welcome, focus on standardization of techniques and transparency of reporting in the biomedical, peer-reviewed literature is commendable. However, that focus has been intermittent as well as lacklustre and so failed to tackle the alarming lack of reliability and reproducibly of biomedical research. Authors have access to numerous recommendations, ranging from simple standards dealing with technical issues to those regulating clinical trials, suggesting that improved reporting guidelines are not the solution. The elemental solution is for editors to require meticulous implementation of their journals’ instructions for authors and reviewers and stipulate that no paper is published without a transparent, complete and accurate materials and methods section.
  
• Impact of New Technologies in Meeting MIQE Guidelines
  New Developments in PCR Instrumentation Help Researchers Meet Modern Regulations
  GEN -- Drug Discovery Tutorial
  15th May 2015 by Andrew Birnie, Ph.D.
  Within any investigation or report submitted for publication, it is crucial to ensure that any process included is undertaken with complete accuracy and reliability. This is true of any scientific technique including but not limited to qPCR.
  As demonstrated by the mistaken link between MMR vaccine and developmental disorders, which led to many children not undergoing vaccination, poor qPCR data can have huge implications. The implementation of the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines, therefore, set out the minimum information necessary for evaluating qPCR experiments.
  However, meeting these guidelines presents its own challenges. Researchers must be able to demonstrate that multiple aspects of their investigation were undertaken correctly and present all the additional relevant data. As well as this, there is a need to show that the instrument being used is up to the necessary standard. All this must be achieved on top of the time and cost restrictions present in any modern laboratory.
• WEBINAR -- Characterizing the Performance of qPCR Instruments – Approaches for Assessment and Comparison
  The breadth of instruments available for quantitative PCR (qPCR) has continued to grow in the past 5-10 years. With older platforms now being retired and an abundance of new technologies available to replace them, lab managers, technicians, and researchers will need to effectively compare and evaluate the performance of these platforms. While new features such as multiplexing, microfluidics, and integration with liquid handling automation have enabled higher throughput and lower operating costs, it has made it increasing complex to readily compare different types of instruments and their respective performance characteristics.
  As the list of features and specifications grows, understanding some of the key metrics of instrument performance will become critical for evaluating platforms that will best meet the needs of a laboratory’s application focus and assay requirements. Unfortunately, instrument vendors have not consistently conformed to any particular standards for defining and assessing performance characteristics of qPCR instruments and rarely have the methods been adequately documented in the product literature.
  In this webinar, we will define several key performance metrics of qPCR instruments such as dynamic range, Cq uniformity, sensitivity, and resolution, and further discuss their importance in practical terms. Using data from characterization and verification studies performed on the IntelliQube instrument from Douglas Scientific, we will also review approaches to evaluating these metrics, including assays and software tools that streamline the analysis and interpretation of performance testing results.
  
• RDML-Ninja and RDMLdb for standardized exchange of qPCR data.
  Ruijter JM, Lefever S, Anckaert J, Hellemans J, Pfaffl MW, Benes V, Bustin SA, Vandesompele J, Untergasser A; and RDML consortium.
  BMC Bioinformatics. 2015 16: 197
  Link to RDML sub-domain
  
  BACKGROUND: The universal qPCR data exchange file format RDML is today well accepted by the scientific community, part of the MIQE guidelines and implemented in many qPCR instruments. With the increased use of RDML new challenges emerge. The flexibility of the RDML format resulted in some implementations that did not meet the expectations of the consortium in the level of support or the use of elements.
  
  RESULTS: In the current RDML version 1.2 the description of the elements was sharpened. The open source editor RDML-Ninja was released ( http://sourceforge.net/projects/qpcr-ninja/ ). RDML-Ninja allows to visualize, edit and validate RDML files and thus clarifies the use of RDML elements. Furthermore RDML-Ninja serves as reference implementation for RDML and enables migration between RDML versions independent of the instrument software. The database RDMLdb will serve as an online repository for RDML files and facilitate the exchange of RDML data ( http://www.rdmldb.org ). Authors can upload their RDML files and reference them in publications by the unique identifier provided by RDMLdb. The MIQE guidelines propose a rich set of information required to document each qPCR run. RDML provides the vehicle to store and maintain this information and current development aims at further integration of MIQE requirements into the RDML format.
  
  CONCLUSIONS: The editor RDML-Ninja and the database RDMLdb enable scientists to evaluate and exchange qPCR data in the instrument-independent RDML format. We are confident that this infrastructure will build the foundation for standardized qPCR data exchange among scientists, research groups, and during publication.
  
• Pre-Congress MIQE Workshop -- MIQE- How to get good quality control in qPCR
  6-7 October 2015 at Metropolitan Hotel Athens, Greece
  20th World Congress on Advances in Oncology and the 18th International Symposium of Molecular Medicine
  
  Are you working with qPCR? Do you have control of the quality in all the steps of the analysis procedure? This course will go deep into the MIQE guidelines, describe the important steps in RNA and DNA analysis with qPCR and how you should work to fulfill the guidelines. To follow the guidelines will give you a better control of the quality of your results. The course is theoretical.
  
  
  

• AGENTS OF CHANGE – MIQE Guidelines New iBook
  by SPLICE 14th May 2015
  Can we trust your published qPCR data?
  The pressure to publish is causing shortcuts which result in misleading papers being published.
  Today there is a great deal of pressure to publish papers in high-ranking, high-impact factor journals in order to make it possible to write successful grant applications. The initial purpose of publishing is overshadowed by the pressure of having as many papers as possible in high-impact factor publications. This has a significant impact on the integrity of the generated data which is too often non-reproducible. Obviously, a paper should enable anyone with the right competencies to repeat what has been done, and the main aim of the paper should be to transmit this information in an understandable way.
• A new Journal -- Biomolecular Detection and Quantification
  by Dale Yuzuki in "Behind the Bench"   22. May 2014
  
  The publisher Elsevier is preparing to launch a new journal called Biomolecular Detection and Quantitation (BDQ) whose goal is “to champion excellence in molecular study design” and a focus on the application of quantitative molecular technologies. BDQ was established by a group of scientists who, in 2009, wrote “The MIQE guidelines: Minimum Information for publication of Quantitative real-time PCR Experiments”, and more recently, in 2013, wrote “The Digital MIQE guidelines: Minimum Information for publication of Quantitative digital PCR Experiments”
• Impact of New Technologies in Meeting MIQE Guidelines
  New Developments in PCR Instrumentation Help Researchers Meet Modern Regulations
  by Andrew Birnie, Ph.D. -- May 15, 2015 (Vol. 35, No. 10)
  Within any investigation or report submitted for publication, it is crucial to ensure that any process included is undertaken with complete accuracy and reliability. This is true of any scientific technique including but not limited to qPCR.
  As demonstrated by the mistaken link between MMR vaccine and developmental disorders, which led to many children not undergoing vaccination, poor qPCR data can have huge implications. The implementation of the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines, therefore, set out the minimum information necessary for evaluating qPCR experiments.
  However, meeting these guidelines presents its own challenges. Researchers must be able to demonstrate that multiple aspects of their investigation were undertaken correctly and present all the additional relevant data. As well as this, there is a need to show that the instrument being used is up to the necessary standard. All this must be achieved on top of the time and cost restrictions present in any modern laboratory.
  
• GUIDING OUR PCR EXPERIMENTS
  Jeffrey Perkel, Ph.D.
  BioTechniques, Vol. 58 (5): 217–221
  Abstract - The MIQE guidelines for qPCR and dPCR have been around for a while now, but few are taking advantage of this resource. Jeffrey Perkel looks at challenge of standardizing PCR.
  
• Policy -- NIH plans to enhance reproducibility.
  Collins FS, Tabak LA
  Nature. 2014 505 (7485): 612-613
  A growing chorus of concern, from scientists and laypeople, contends that the complex system for ensuring the reproducibility of biomedical research is failing and is in need of restructuring. As leaders of the US National Institutes of Health (NIH), we share this concern and here explore some of the significant interventions that we are planning.
  Science has long been regarded as ‘self-correcting’, given that it is founded on the replication of earlier work. Over the long term, that principle remains true. In the shorter term, however, the checks and balances that once ensured scientific fidelity have been hobbled. This has compromised the ability of today’s researchers to reproduce others’ findings.
  Let’s be clear: with rare exceptions, we have no evidence to suggest that irreproducibility is caused by scientific misconduct. In 2011, the Office of Research Integrity of the US Department of Health and Human Services pursued only 12 such cases. Even if this represents only a fraction of the actual problem, fraudulent papers are vastly outnumbered by the hundreds of thousands published each year in good faith.
  Instead, a complex array of other factors seems to have contributed to the lack of reproducibility. Factors include poor training of researchers in experimental design; increased emphasis on making provocative statements rather than presenting technical details; and publications that do not report basic elements of experimental design. Crucial experimental design elements that are all too frequently ignored include blinding, randomization, replication, sample-size calculation and the effect of sex differences. And some scientists reputedly use a ‘secret sauce’ to make their experiments work — and withhold details from publication or describe them only vaguely to retain a competitive edge. What hope is there that other scientists will be able to build on such work to further biomedical progress?
  Exacerbating this situation are the policies and attitudes of funding agencies, academic centres and scientific publishers. Funding agencies often uncritically encourage the overvaluation of research published in high-profile journals. Some academic centres also provide incentives for publications in such journals, including promotion and tenure, and in extreme circumstances, cash rewards.
  Then there is the problem of what is not published. There are few venues for researchers to publish negative data or papers that point out scientific flaws in previously published work. Further compounding the problem is the difficulty of accessing unpublished data — and the failure of funding agencies to establish or enforce policies that insist on data access.
• EDITORIAL -- Reproducibility
  Marcia McNutt (Editor-in-Chief of Science)
  Science advances on a foundation of trusted discoveries. Reproducing an experiment is one important approach that scientists use to gain confidence in their conclusions. Recently, the scientific community was shaken by reports that a troubling proportion of peer-reviewed preclinical studies are not reproducible. Because confidence in results is of paramount importance to the broad scientific community, we are announcing new initiatives to increase confidence in the studies published in Science. For preclinical studies (one of the targets of recent concern), we will be adopting recommendations of the U.S. National Institute of Neurological Disorders and Stroke (NINDS) for increasing transparency.* Authors will indicate whether there was a pre-experimental plan for data handling (such as how to deal with outliers), whether they conducted a sample size estimation to ensure a sufficient signal-to-noise ratio, whether samples were treated randomly, and whether the experimenter was blind to the conduct of the experiment. These criteria will be included in our author guidelines.
• Journals unite for reproducibility
  NATURE | EDITORIAL 05 November 2014
  Consensus on reporting principles aims to improve quality control in biomedical research and encourage public trust in science.
  
• Embracing minimal guidelines for the reporting of RT-qPCR experiments: responsibility lies on both ends
  Correspondence:  Alejandro Montenegro-Montero, Stephen A. Bustin and Jo Vandesompele
  In mid-2012, Stephen A. Bustin, Jo Vandesompele and Alejandro Montenegro-Montero, decided to send a letter to the editor of a glam magazine asking for journals to demand authors to provide at least minimal information for the critical evaluation and reproducibility of published RT-qPCR experiments. The lack of information regarding these experiments is inversely proportional to the IF of the journal: the higher the IF, the lower the amount of information provided for these experiments (See Nat Methods. 2013 Nov;10(11):1063-7). It was no surprise then, considering that they were the ones we targeted in the letter (although not explicitly), that glam journals (you know which…) refused publishing the letter. I found the letter searching for something else in my computer and decided to share it with you, just as it was written back in 2012. The main theme is as true as it was back then.
  
• From benchtop to desktop: important considerations when designing amplicon sequencing workflows.
  by Murray DC, Coghlan ML1, Bunce M
  PLoS One. 2015 10(4): e0124671
  Amplicon sequencing has been the method of choice in many high-throughput DNA sequencing (HTS) applications. To date there has been a heavy focus on the means by which to analyse the burgeoning amount of data afforded by HTS. In contrast, there has been a distinct lack of attention paid to considerations surrounding the importance of sample preparation and the fidelity of library generation. No amount of high-end bioinformatics can compensate for poorly prepared samples and it is therefore imperative that careful attention is given to sample preparation and library generation within workflows, especially those involving multiple PCR steps. This paper redresses this imbalance by focusing on aspects pertaining to the benchtop within typical amplicon workflows: sample screening, the target region, and library generation. Empirical data is provided to illustrate the scope of the problem. Lastly, the impact of various data analysis parameters is also investigated in the context of how the data was initially generated. It is hoped this paper may serve to highlight the importance of pre-analysis workflows in achieving meaningful, future-proof data that can be analysed appropriately. As amplicon sequencing gains traction in a variety of diagnostic applications from forensics to environmental DNA (eDNA) it is paramount workflows and analytics are both fit for purpose.
  
• Deliver verifiable qPCR results using MIQE guidelines and automation
  Modern advances in molecular diagnostics and personalized medicine rely on technologies that accurately detect risk, diagnose disease, and monitor therapies that will work best in individual patients. One such molecular diagnostic technique is reverse transcription followed by real-time quantitative PCR (RT-qPCR), which is used to quantitate mRNA and microRNA differences in gene expression levels between samples. Inadequate sample and nucleic acid preparation, improper qPCR assay design, and faulty data and statistical analyses can lead to unreliable and erroneous results.
  In this webinar we will introduce strategies and quality management techniques for standardizing RT-qPCR experiments. In particular, incorporating the ‘Minimum Information for Publication of Quantitative Real-Time PCR Experiments’ (MIQE) guidelines into molecular diagnostic protocols and analytical workflows. We will also review several key considerations when optimizing RT-qPCR experiments, e.g. the source of biological sample, quality of extracted RNA, selection of the correct reference genes, and application of the correct calculations or statistics when reporting results. We will discuss how these approaches, combined with automation, will limit experimental error and improve sample traceability in generating unequivocal, verifiable results.
• Optimisation, MIQE & QC strategies in qPCR -- Session at the qPCR & NGS 2015 Symposium
  Time:  Wednesday, 25/Mar/2015:  8:30am - 12:30pm
  Location: Lecture hall 14
  650 participants, TUM Weihenstephan http://www.qPCR-NGS-2015.net
  Session Chair: Tania Nolan, The Gene Team, United Kingdom
  Session Chair: Afif M. Abdel Nour, Bio-Rad, United Arab Emirates
  
• The Top 5 Most Cited Articles on Molecular Diagnostics
  Clinical Chemistry's January 2015 issue is dedicated to Molecular Diagnostics. Drs. Rossa W.K. Chiu, Frank R. Cockerill, Y.M. Dennis Lo, and Carl T. Wittwer will highlight recent advances in molecular diagnostics that focus on clinical applications that use molecular diagnostics to reach novel conclusions about disease and therapy and on new technologies that improve test turnaround time, high-volume needs, comprehensive analysis, or ease of use.
  In view of this exciting Molecular Diagnostics Special Issue in January, find here the mostly highly cited Clinical Chemistry articles focused on molecular diagnostics since 2009.
  
1. 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
   The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments
   Volume 55 Issue 4: April 2009
   
2. Jessica A. Weber, David H. Baxter, Shile Zhang, David Y. Huang, Kuo How Huang, Ming Jen Lee, David J. Galas, and Kai Wang
   The MicroRNA Spectrum in 12 Body Fluids
   Volume 56 Issue 11: November 2010
   
3. Xu Ji, Rie Takahashi, Yumiko Hiura, Go Hirokawa, Yasue Fukushima, and Naoharu Iwai
   Plasma miR-208 as a Biomarker of Myocardial Injury
   Volume 55 Issue 11: November 2009
   
4. Yi Zhang, Yin Jia, Ruiying Zheng, Yingjun Guo, Yue Wang, Hui Guo, Mingyu Fei, and Shuhan Sun
   Plasma MicroRNA-122 as a Biomarker for Viral-, Alcohol-, and Chemical-Related Hepatic Diseases
   Volume 56 Issue 12: December 2010
   
5. Taichi Adachi, Michio Nakanishi, Yoritaka Otsuka, Kunihiro Nishimura, Gou Hirokawa, Yoichi Goto, Hiroshi Nonogi, and Naoharu Iwai
   Plasma MicroRNA 499 as a Biomarker of Acute Myocardial Infarction
   Volume 56 Issue 7: July 2010
• A new Journal -- Biomolecular Detection and Quantification
  Gepostet von Dale Yuzuki in Behind the Bench
  The publisher Elsevier is preparing to launch a new journal called Biomolecular Detection and Quantitation (BDQ) whose goal is “to champion excellence in molecular study design” and a focus on the application of quantitative molecular technologies. BDQ was established by a group of scientists who, in 2009, wrote “The MIQE guidelines: Minimum Information for publication of Quantitative real-time PCR Experiments”, and more recently, in 2013, wrote “The Digital MIQE guidelines: Minimum Information for publication of Quantitative digital PCR Experiments”.
  

2014

• The reproducibility of biomedical research: Sleepers awake!
  Stephen A. Bustin,
  Biomolecular Detection and Quantification, Volume 2, December 2014, Pages 35–42
  There is increasing concern about the reliability of biomedical research, with recent articles suggesting that up to 85% of research funding is wasted. This article argues that an important reason for this is the inappropriate use of molecular techniques, particularly in the field of RNA biomarkers, coupled with a tendency to exaggerate the importance of research findings.
• Prime time for qPCR – raising the quality bar
  by Mikael Kubista
  Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific
  technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is
  preferred in research applications, it has still to win broad acceptance in routine. Main hurdles are the lack of
  guidelines, standards, quality controls, and even proper methods to evaluate the diagnostic results. This is now
  rapidly changing.
  
• Next Steps in Reproducibility
  By Damian Pattinson and Virginia Barbour Posted: November 13, 2014
  In last week’s Nature and Science, the outcome of a meeting convened by NIH, Nature, and Science to discuss the issue of lack of reproducibility in the basic science research literature was published. This meeting, which brought together representatives from publishers (including PLOS), and many representatives from the NIH and other funders, produced a series of principles, Proposed Principles and Guidelines for Reporting Preclinical Research, which were endorsed by a large and diverse group of publishers, associations, and societies including ourselves. The main principles are as follows:
  - Rigorous statistical analysis
  - Transparency in reporting
  - Data and material sharing
  - Consideration of refutations
  - Consider establishing best practice guidelines for image based data and descriptions of biological data.
  Read more -...
  
• Keeping qRT-PCR rigorous and biologically relevant.
  Bennett J, Hondred D, Register JC 3rd. in Plant Cell Rep. 2014 Oct 11.
  MIQE has received support from journals, authors and suppliers of equipment, reagents, and software (http://miqepress.gene-quantification.info/). De Keyser et al. (2013) and Saha and Blumwald (2014) proved that qRT-PCR data can be rigorously conducted and reported without inclusion of a MIQE checklist. However, we do advocate that the items on the checklist be addressed during experimental design and execution and recommend that a checklist be made available to referees during the review process. Except for rare and justified cases (Bustin et al. 2010), reference genes should be published in conjunction with their use in normalizing target genes within a biological context. Finally, we emphasize that, for every manuscript that includes qRT-PCR data, it is vital that authors give careful attention to the qRT-PCR experimental details to ensure that gene expression data from this powerful method are valid.
• An evidence based strategy for normalization of quantitative PCR data from miRNA expression analysis in forensically relevant body fluids.
  Sauer E, Madea B, Courts C. in Forensic Sci Int Genet. 2014 11: 174-181
  An evidence based strategy for normalization of quantitative PCR data from miRNA expression analysis in forensic organ tissue identification.
  Sauer E, Babion I, Madea B, Courts C. inForensic Sci Int Genet. 2014 13: 217-223
• Study Shows High Variability in Reverse Transcription Efficiency
  Nov 13, 2014 via GenomeWeb
  http://www.clinchem.org/content/early/2014/10/28/clinchem.2014.230615.abstract
  Though reverse transcription of RNA to cDNA is an essential first step for a growing number of genomics applications, researchers have long known of problems associated with RT and its effects on study results. Now, a new study has examined some of those issues and has provided a workflow and guidelines for researchers publishing RT-based data.
  The study, published in Clinical Chemistry late last month, was undertaken by some of the authors of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments, or MIQE, guidelines, which debuted in 2009. It compared measures of different mRNA targets using six commercially available RT enzymes and varying sample concentrations of differing qualities. Results showed the variability was "sufficiently large to call into question the validity of many published data that rely on quantification of cDNA."  ... ...
  
• Sigma Aldrich Learning Center -- qPCR and MIQE Seminar Series
  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.
  - See more at: http://www.sigmaaldrich.com/life-science/learning-center/customer-education/qpcr-miqe-seminar-series.html
• MIQE Guidelines for Digital PCR
  by Sylvia Norman on 4th of September, 2014 in PCR & Real-time PCR
  MIQE what’s that?
  When writing dPCR materials and methods for a paper have you ever pondered what information you should include? This is where the MIQE guidelines will really help. Guidelines for minimum information required for publication of a digital PCR (dPCR) experiment were published by JF Huggett et al. in 2013. These were a follow-up to minimum information requirements for publication of real-time PCR experiments (qPCR) suggested by SA Bustin et al. in 2009.  MIQE guidelines for qPCR and dPCR publications are divided into the following categories:  essential information and desirable information. Essential Information must be included in the submitted manuscript or accompanying supplemental material. Desirable Information should be included in the submitted manuscript and is intended to help the reader understand the study.
  
• To do's and dont's
  The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE)
  Taylor SC and Mrkusich EM;  J Mol Microbiol Biotechnol. 2014 24(1): 46-52
  
  In the past decade, the techniques of quantitative PCR (qPCR) and reverse transcription (RT)-qPCR have become accessible to virtually all research labs, producing valuable data for peer-reviewed publications and supporting exciting research conclusions. However, the experimental design and validation processes applied to the associated projects are the result of historical biases adopted by individual labs that have evolved and changed since the inception of the techniques and associated technologies. This has resulted in wide variability in the quality, reproducibility and interpretability of published data as a direct result of how each lab has designed their RT-qPCR experiments. The 'minimum information for the publication of quantitative real-time PCR experiments' (MIQE) was published to provide the scientific community with a consistent workflow and key considerations to perform qPCR experiments. We use specific examples to highlight the serious negative ramifications for data quality when the MIQE guidelines are not applied and include a summary of good and poor practices for RT-qPCR.

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.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.
  

• 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

• 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
  
  • Read about MIQE and qPCR quality concerns in Nature Methods (2013) 10
    
  • Info on TATAA qPCR QC products
• 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

• 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.

• 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
  
  
  • MIQE, guidelines for minimum information required for publication of qPCR data have been published in Clinical Chemistry.
  • A concise list of MIQE requirements for BMC journals has been published
  • MIQE police with regards to primer sequence disclosure has been clarified
  • A MIQE website has been launched
  
  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
  May 19, 2011  by  Bio-Rad
  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)

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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