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Dear researcher,
dear Gene Quantification page reader,
Our newsletter informs about the latest news in quantitative real-time
PCR (qPCR and qRT-PCR), which are compiled and summarised on the Gene
Quantification homepage. The focus of this newsletter
issue is:
- MIQE guidelines
- new paper => http://miqe.gene-quantification.info
- latest press review => http://miqe-press.gene-quantification.info/
- qPCR 2010 Event in Vienna => final announcement
- qPCR Application Workshops - and more ... ... ...
The MIQE
Guidelines Minimum Information for Publication of Quantitative
Real-Time PCR Experiments
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, & Carl T. Wittwer
Clinical Chemistry 2009, 55(4): 611-622
BACKGROUND:
Currently, a lack of consensus exists on how best to perform and
interpret quantitative real-time PCR (qPCR) experiments. The
problem is exacerbated by a lack of sufficient experimental detail in
many publications, which impedes a reader's ability to evaluate
critically the quality of the results presented or to repeat the
experiments.
CONTENT: The
Minimum Information for Publication of Quantitative Real-Time PCR
Experiments (MIQE) guidelines target the reliability of results to help
ensure the integrity of the scientific literature, promote consistency
between laboratories, and increase experimental transparency. MIQE is a
set of guidelines that describe the minimum information necessary for
evaluating qPCR experiments. Included is a checklist to accompany the
initial submission of a manuscript to the publisher. By providing all
relevant experimental conditions and assay characteristics, reviewers
can 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.
SUMMARY: Following these
guidelines will encourage better experimental practice, allowing more
reliable and unequivocal interpretation of qPCR results. http://miqe.gene-quantification.info
Why
the need for
qPCR publication guidelines? - The case for MIQE
Stephen A.
Bustin
Methods. 2010 - in qPCR special issue - The ongoing
evolution of qPCR
Institute of Cell and
Molecular Science, Barts and the London School of Medicine and
Dentistry, Queen Mary University of London, London, UK
The polymerase chain reaction (PCR) has matured from a labour- and time-intensive, low throughput qualitative gel-based technique to an easily automated, rapid, high throughput quantitative technology. Real-time quantitative PCR (qPCR) has become the benchmark technology for the detection and quantification of nucleic acids in a research, diagnostic, forensic and biotechnology setting. However, ill-assorted pre-assay conditions, poor assay design and inappropriate data analysis methodologies have resulted in the recurrent publication of data that are at best inconsistent and at worst irrelevant and even misleading. Furthermore, there is a lamentable lack of transparency of reporting, with the "Materials and Methods" sections of many publications, especially those with high impact factors, not fit for the purpose of evaluating the quality of any reported qPCR data. This poses a challenge to the integrity of the scientific literature, with serious consequences not just for basic research, but potentially calamitous implications for drug development and disease monitoring. These issues are being addressed by a set of guidelines that propose a minimum standard for the provision of information for qPCR experiments ("MIQE"). MIQE aims to restructure to-day's free-for-all qPCR methods into a more consistent format that will encourage detailed auditing of experimental detail, data analysis and reporting principles. General implementation of these guidelines is an important requisite for the maturing of qPCR into a robust, accurate and reliable nucleic acid quantification technology.
RDML:
structured language and reporting guidelines for real-time quantitative
PCR data
Lefever S,
Hellemans J, Pattyn F, Przybylski DR, Taylor C, Geurts R, Untergasser
A, Vandesompele J; on behalf of the RDML consortium. Center for Medical
Genetics, Ghent University Hospital, Ghent, Belgium.
Nucleic Acids Res. 2009 Apr;37(7):
2065-2069
The XML-based Real-Time PCR Data Markup Language (RDML) has been developed by the RDML consortium (http://www.rdml.org) to enable straightforward exchange of qPCR data and related information between qPCR instruments and third party data analysis software, between colleagues and collaborators and between experimenters and journals or public repositories. We here also propose data related guidelines as a subset of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to guarantee inclusion of key data information when reporting experimental results. http://rdml.gene-quantification.info/
Reliability
of real-time reverse-transcription PCR in clinical diagnostics:
gold standard or sub-standard?
Murphy J,
Bustin SA.
Expert
Rev Mol Diagn. 2009 9(2):187-197
Unreliable
real-time PCR analysis of human endogenous retrovirus-W (HERV-W) RNA
expression and DNA copy number in multiple sclerosis.
Garson JA,
Huggett JF, Bustin SA, Pfaffl MW, Benes V, Vandesompele J, Shipley GL.
AIDS
Res Hum Retroviruses. 2009 25(3): 377-378
Real-time
polymerasechain reaction – towardsa more reliable, accurateand relevant
assay.
SA Bustin
EUROPEAN
PHARMACEUTICAL REVIEW 2008 (6): 19-27
In-House
Nucleic Acid Amplification Assays in Research: How Much Quality
ControlIs Needed before One Can Rely upon the Results?
Petra
Apfalter, Udo Reischl and Margaret R. Hammerschlag
JOURNAL
OF CLINICAL MICROBIOLOGY 2005 (12): 5835–5841
The latest news from our MIQE press review => http://miqe-press.gene-quantification.info/
- Real-time PCR
on SciTopics
UPDATE on 21 January 2010 by Prof Stephen Bustin
Category: Biochemistry, Genetics and Molecular Biology
Guidelines for minimum information required for publication of qPCR data have now been published in Clinical Chemistry - qPCR Assay Quality
Assessment on SciTopics
UPDATE on 21 January 2010 by Prof Stephen Bustin
Category: Biochemistry, Genetics and Molecular Biology
Guidelines for minimum information required for publication of qPCR data have now been published in Clinical Chemistry - November 11, 2009
- Videos explaining MIQE guidelines
Browsing through You Tube just now, I found these videos illustrating the concepts of the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. These focus on how to apply the guidelines to design a solid experimental approach for RT-qPCR. There are four videos in total. The sound is a bit “fuzzy,” but the content is a fairly nice overview of MIQE. - Helixis Tutorial: MIQE guidelines: a bench perspective on
use and benefits
Description: "MIQE guidelines from a scientist perspective and discussion on their use and benefits when performing Real-Time PCR experiments. "
Total Running Time: 9:52 (posted 10/29/2009) Direct YouTube link => http://www.youtube.com/watch?v=zm9QoIpOzkM - MIQE checklist - http://www.helixis.com/support/usefultools/MIQE_Checklist.pdf
Description: To help you follow the latest MIQE guidelines, Helixis has formatted this useful checklist to keep handy at your bench or desk when designing your Real-Time PCR experiments or drafting your next paper. - Do Your RT-qPCRs Make The Grade?
26th July 2009 - Real-time PCR is a technique that is now commonly employed in almost all molecular biology laboratories to quickly answer very specific questions. Northern and Southern blotting are now a thing of the past. No longer do we wait days to know whether a gene is expressed. We can have the answer in 45 minutes!
But with the widespread use of such a wonderful and sensitive technology comes differences in how results are reported in the literature. There are also differences between reviewers reading these papers and their understanding of the essential information required to judge the accuracy of the reported data.
To overcome this increasing problem of lack of consistency in publications, a panel of real-time PCR experts published a set of guidelines containing what they consider the minimal information required when reporting qPCR results. That paper called The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, was published February 2009 in the Journal of Clinical Chemistry. - Publishing Data That Conform to the
MIQE Guidelines
Minimum information for publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines help researchers design qPCR experiments.
Real-time quantitative polymerase chain reaction (qPCR) is a definitive technique for quantifying differences in gene expression levels between samples. However, a lack of consistency in experimental design and reporting combined with inadequate guidelines to review submitted articles with qPCR data greatly increases the potential of reporting statistically insignificant and conflicting results.1 The publication2 and retraction3 of a Science “Breakthrough of the Year 2005” article underlines the issue. - MIQE Guidelines 'Slowly Filtering
Through' PCR Community Despite Lack of Journal Enforcement
by Bernadette Toner Genome Web
Guidelines proposed in early 2009 to help standardize how qPCR results are reported are "slowly filtering through" the research community, but much work still needs to be done to improve the quality of published qPCR studies, according to one of the authors of the standard. - Are your qPCR experiments compliant
with MIQE?
The MIQE guidelines establish specifications for the minimum information that must be reported for a qPCR experiment in order to ensure its relevance, accuracy, correct interpretation and repeatability. Comply with MIQE guidelines ! Learn why Prof. Kubista from the TATAA Biocenter uses the Agilent 2100 Bioanalyzer for RNA quality control => Start webinar - Feature Article - PCR
Technology Review: Standardization of qPCR and RT-qPCR - New Guidelines
Seek to Promote Accurate Interpretation of Data and Reliable
Results by Stephen A. Bustin, Jo Vandesompele, Michael W. Pfaffl => download PDF
The perceived ease of use of real-time quantitative PCR (qPCR) and reverse transcription PCR (RT-qPCR) technology has revolutionized life science research. Its effectiveness at amplification and quantification of low levels of nucleic acids has driven the emergence of numerous applications, including cellular mRNA and miRNA quantification, biomarker discovery and validation, microbial quantification, cancer risk assessment, gene dosage determination, and detection of extremely low copy targets for forensic investigations. This, in turn, has resulted in an abundance of publications utilizing qPCR data obtained with diverse reagents, protocols, analysis methods, and reporting formats. Unfortunately, few papers report in detail how these results were obtained. This lack of clarity and transparency has led to concern in the research community over the reliability of qPCR data interpretation and the real danger of the scientific literature being corrupted with publications reporting erroneous and conflicting results. This has already occurred in some cases, resulting, for example, in retraction of a Science “Breakthrough of the Year 2005” report. Now that qPCR has come of age, standardization is needed to ensure its validity, prompting the recent formulation of guidelines to increase experimental transparency, promote consistency between laboratories, and therefore, help assure the publication of valid conclusions. - A practical approach to RT-qPCR - Publishing
data that conforms to the MIQE guidelines
(Bio-Rad amplification tech note 5859) by Sean Taylor et al., Bio-Rad Laboratories, Hercules, CA - MIQE Guidelines - RNA Qualitätskontrolle in
der Genexpressionsanalytik – ein Meilenstein auf dem Weg zum
Erfolg (in German)
by Christiane Becker, Irmgard Riedmaier, and Michael W. Pfaffl
Abstrakt (D) - Die Qualität des Probenmaterials, also der Gesamt-RNA, hat einen markanten Einfluss auf die Richtigkeit der quantitativen RT-PCR. Die Überprüfung der RNA Qualität vor einer Expressionsmessung ist unabdingbar, um verlässliche RT-qPCR Expressionsergebnisse zu erhalten.
Abstract (E) - The integrity of total RNA has a distinct influence on the accuracy of RT-qPCR. Quality assessment is an essential step for the evaluation of reliable results in gene expression analysis.
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qPCR 2010 in Vienna International qPCR Symposium & Exhibition 7-9th April 2010 http://www.qpcr2010-vienna.net/ Main Topic “The ongoing evolution of qPCR” |
The great international interest of the previous International qPCR Events from 2004 to 2009 with up to 600 participants from over 40 countries, and 32 international companies in the Exhibition motivates repeating the success next year in Vienna - qPCR 2010 Vienna international symposium 7th – 9th April 2010. The focus of the qPCR 2010 Event will be “The ongoing evolution of qPCR” representing all new and emerging techniques, applications and data analysis methods: MIQE Gudelines, HRM, microRNA, CNV, single-cell qPCR, digital PCR, and analysis of circulating nucleic acids will be in the focus of the conference. The talks topics by the keynote lecturer and selected invited academic speakers will be published in a METHODS special qPCR issue with the title “The ongoing evolution of qPCR”, at the meeting in cooperation with Elsevier.
Why Vienna? The Vienna region is Austria’s largest life sciences location. The Austrian government and the City of Vienna set up a biotechnology network (LISA VR www.lisavr.at) with the aim of having one central body to provide support and advice to start-ups and high-tech companies in the biotec field. Currently 175 life science companies employ 11,000 people in the greater Vienna region. In addition an estimated 4,500 academic researchers work in the life science sector. Vienna is the economic and biotechnology hub for Central and Eastern Europe and has become an innovative center for the life science in recent years.
The
event is structured in two parts:
1) an International qPCR Symposium taking place
April 7 - 9, including various talk sessions in
the big lecture hall fitting 350 persons,
2) a parallel qPCR Industrial Exhibition with
40 companies in the Aula and the Basement of the Juridicum (Juridicum
der Universität Wien) directly in the city center of Vienna.
Please register now => http://registration.qPCR2010-Vienna.net
qPCR
Symposium topic - “The ongoing
evolution of qPCR”
The symposium focus will be on 40 lectures presented by international
recognised experts in their application fields. The emphasis will be on
unbiased, didactic information exchange. The focus of the qPCR 2010 Event will be “The ongoing evolution of qPCR”
representing all new and emerging techniques, applications
and data analysis methods:
Symposium sessions:
MIQE and QM
strategies in qPCR
The MIQE guidelines: minimum information for
publication of quantitative real-time PCR experiments. Following these
guidelines will encourage better experimental practice, allowing more
reliable and unequivocal interpretation of qPCR results. QM strategies
in real-time PCR to guarantee better and more valid results.
High throughput
quantitative PCR – digital PCR
384 and 1536 well applications, new high
throughput platforms, droplet PCR, qPCR robotics, digital PCR, gene
expression real-time RT-PCR arrays (mRNA and microRNA), quantitative
multiplexing, …
HRM – High
Resolution Melting - Epigenetics
SNP analysis, HRM = high resolution melt
applications, Epigenetics, methylation markers, HRM platform
comparison, …
CNA -
Circulating nucleic acids
Analysis of circulating RNAs and DNA and microRNAs
as diagnostic and prognostic marker, …
Single-cell
qPCR
Single-cell sampling, pre-amplification
techniques, laser microdissection, sub-cellular PCR, micro-manipulation
of cell clusters, cellular micro injection, FACS spotting, single cell
handling, pre-amplification, …
RNAi - microRNA -
siRNA Applications – miRNA normalisation
RNAi mechanism, microRNA extraction, qRT-PCR
technologies to detect microRNA, microRNA normalisation strategies,
siRNA applications in combination with qRT-PCR, microRNA targets and
microRNA precursors, new siRNA manipulation and microRNA technologies,
...
qPCR BioStatistics
& BioInformatics
software applications, data mining, calculation of
relative expression, primer and probe design on mRNA and microRNA
level, real-time PCR efficiency determination, mathematical modelling,
multivariate expression profiling, statistics in real-time PCR, data
management, multiway expression profiling, multiple regression
analysis, 3D data visualization, ...
Register now => http://registration.qPCR2010-Vienna.net
workshops
BioEPS GmbH - qPCR Application Workshops
Life Science is still a growing
sector and new methods and technologies are continously developed.
Therefore permanent training and education becomes so important.
With our specific course program we are offering a range of
high-quality course modules to give a general and independent
overview of existing qPCR technologies and systems. Our course issues
are based on skilled know-how from own research studies and
publications.
Our aim is to point out a critical way of thinking to increase the
quality and outcome of experimental data.
All courses are
held regularly in Freising-Weihenstephan, Germany, in German and
English language. Further customized
workshops and specialized trainings will be held as well across Europe
and world-wide. Workshops are powered by BioEPS
GmbH, located at the campus of the
Technical University of Munich, in Freising-Weihenstephan, very close
to the Munich Airport (MUC).
For more information and
registration, please see our web page => http://workshops.gene-quantification.info
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Course modules 2010:
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Course dates 2010:
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Best regards,
Michael W. Pfaffl
responsible Editor of the Gene
Quantification Pages
http://www.gene-quantification.info
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