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 MIQE qPCR APP for iPhone, iPad and iPod -
iOS Universal
New version available - 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 compliance to
MIQE in minutes, have all required references to hand, and follow qPCR
events and news ... ... slide show
Over 6,500 downloads => http://itunes.apple.com/app/miqe-qpcr/id423650002?mt=8
MIQE qPCR - Best Of The Web: Feb 2012 (Vol.
32, No. 3)
Rated
* * * => VERY GOOD => Nice user interface, strong reference
resource
rated by Genetic Engineering News |
|
online since
November 2011
|
|
ANDROID MARKET MIQE qPCR - 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 compliance to
MIQE in minutes, have all required references to hand, and follow qPCR
events and news.
- 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.
- Suggestions collected from experts at the qPCR
2011 symposium in Freising were included.
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Since May 2012 a
html5 version of the
MIQE_qPCR APP is online
=> MIQE-html5.gene-quantification.info
To run this application you need a html5 compatible web browser!
|
2013
- MIQE: Guidelines for the Design and
Publication of a Reliable Real-time PCR Assay
from Jim Huggett, Tania Nolan and Stephen A. Bustin writing in
Real-Time PCR: Advanced Technologies and Applications:
The capacity to amplify and detect trace amounts of nucleic acids has
made the polymerase chain reaction (PCR) the most formidable molecular
technology in use today. Its versatility and scope was further
broadened first with the development of reverse transcription (RT)-PCR,
which opened up the entire RNA field to thorough exploration and then,
most conspicuously, with its evolution into real-time quantitative PCR
(qPCR). Speed, simplicity, specificity, wide linear dynamic range,
multiplexing and high throughput potential, reduced contamination risk,
simplified detection and data analysis procedures as well as
availability of increasingly affordable instrumentation and reduced
reagent cost have made qPCR the molecular method of choice when
quantifying nucleic acids. Detection of pathogens, SNP analyses and
quantification of RNA, even real-time analysis of gene expression in
vivo have become routine applications and constant enhancements of
chemistries, enzymes, mastermixes and instruments continue to extend
the scope of qPCR technology by promising added benefits such as
extremely short assay times measured in minutes, low reagent usage and
exceptionally rapid heating/cooling rates. The whole process is driven
by the insatiable demand for ever-more specific, sensitive, convenient
and cost-effective protocols. However, it has also become clear that
variable pre-assay conditions, poor assay design and incorrect data
analysis have resulted in the regular publication of data that are
often inconsistent, inaccurate and often simply wrong. The problem is
exacerbated by a lack of transparency of reporting, with the details of
technical information wholly inadequate for the purpose of assessing
the validity of reported qPCR data. This has serious consequences for
basic research, reducing the potential for translating findings into
valuable applications and potentially devastating implications for
clinical practice. In response, guidelines proposing a minimum standard
for the provision of information for qPCR experiments ("MIQE") have
been launched. These aim to establish a standard for accurate and
reliable qPCR experimental design as well as recommendations to ensure
comprehensive reporting of technical detail, indispensable conditions
for the maturing of qPCR into a robust, accurate and reliable nucleic
acid quantification technology.
- MIQE and qPCR quality concerns
2nd May 2013 by TATAA Biocenter
- MIQE qPCR Guidelines -- Three Years Later
05/01/2013 in Biotechniques by Lauren Arcuri Ware
Three years ago, a group of researchers began campaigning for the
adoption of guidelines that promised to improve the reproducibility of
RT-qPCR data. Lauren Arcuri Ware reports on the adoption and evolution
of these guidelines.
- 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
- 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
- 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
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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
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MIQE Guidelines
slowly entering "high impact" journals! |
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LIFE SCIENCE TECHNOLOGIES - qPCR
Innovations and Blueprints
7. October 2011 by Chris Tachibana in ScienceMag.org PDF version
Quantitative PCR users can rapidly generate large amounts of
high-quality data with new instruments and products made possible by
microfluidics and miniaturization technology. These platforms are the
tools for developing techniques that require extremely high throughput
and sensitivity such as digital PCR and single-cell analysis.
Researchers are adopting these methods to ask sophisticated questions
about genetics and cancer biology as well as to develop novel research
and diagnostic assays. As qPCR innovators explore new frontiers and
everyday users venture into more complicated workflows, international
groups of industry and academic partners are keeping us on the path of
best practices. Two consortia (MIQE
& SPIDIA) are generating guidelines on the qPCR
process - from experimental design and pre-analysis sample collection,
to
processing data and publishing results. The guidelines are blueprints
that ensure reproducibility, validity, and transparency.
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LIFE
SCIENCE TECHNOLOGIES - Gene-Expression Analysis Exploits More
Technologies
Science - November 2011 PDF version
To quantify the expression of specific genes, researchers can use a
variety of techniques, including arrays, PCR, and high throughput
sequencing. However, getting accurate results still depends on
precisely carrying out these methods, even with increasingly
user-friendly technologies. In fact, as more scientists study gene
expression, the standards for analysis are growing more rigorous to
ensure that only accurate data are published. Likewise, software has
been keeping pace, helping researchers follow protocols and analyze
their results.
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qPCR
- quicker and easier but don't be sloppy
by Monya
Baker Nature Methods 8, 207–212 (2011) PDF version
Gene profiling using quantitative PCR is becoming
higher throughput,
but researchers must be careful in gathering their data.
Stephen Bustin knew something was wrong as soon as he visited the
laboratory. He was investigating reports that using a technique called
real-time quantitative PCR (qPCR) researchers had identified measles
virus in intestinal tissue of children with developmental disorders1.
If true, those results supported the theory that a commonly
administered vaccine caused autism in young children. If not, anxieties
of parents and public health officials had been needlessly inflamed.
Bustin found that this laboratory was next door to a facility producing
DNA plasmids - a likely source of contamination. Even worse, on at
least two occasions the researchers had neglected a basic step. Because
measles virus is made of RNA, it must be converted to DNA before PCR
can work. The enzyme that effects this conversion, reverse
transcriptase, had been left out of some protocols, but there was no
change in the results. Whatever they had detected was certainly not
measles virus.
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Routine
lab method's accuracy called into question
Nature Medicine Vol 16, page 349
(2010)
by Catherine Shaffer in Nature
Medicine PDF version
In 2002, four years
after
first sparking public controversy over whether the measles, mumps and
rubella vaccine causes autism, Andrew Wakefield reported a possible
molecular mechanism for the connection. He claimed that a form of
irritable bowel disease, which he called autistic enterocolitis, was
triggered by the measles virus (Molec. Pathol. 55, 84–90, 2002). That
finding, however, was based on a “defective experimental technique,”
Stephen Bustin, a molecular biologist at Barts and the London School of
Medicine and Dentistry, told a US federal court in 2007. The problem:
Wakefield had incorrectly applied the common laboratory protocol known
as quantitative real-time polymerase chain reaction (qPCR) to come to
his conclusions.
Bustin says this faulty lab work is a problem shared by many
researchers around the world who have turned to qPCR to measure gene
expression. Unlike standard PCR, which can only crudely quantify levels
of DNA, the chemistry behind qPCR allows researchers to assess such
levels more precisely by comparing sequences of interest against a
known reference added to the test tube mix as a control.
But the reference genes used in qPCR can vary between experiments and
laboratories, which can give misleading results or make it difficult to
compare one study to another. As a result of this and other variables
in the technique, a majority of scientific papers involving qPCR
include flawed methods, say a team of leading qPCR experts. Most
qPCR methods, as reported in the literature, are improperly validated
and irreproducible, Bustin claims.
Last year, he and 11 colleagues published a set of more than 60
individual standards - collectively called the Minimum Information for Publication of Quantitative
Real-Time PCR Experiments (MIQE) to address this problem ( Clin. Chem.
55, 611–622, 2009 ). “If you look at the literature, you find
again and again and again the appalling quality of qPCR protocols,”
says Bustin, who this month repeated his call for the scientific
community to adopt the MIQE guidelines (Methods 50, 217–226, 2010).
“There's no excuse for anyone either not reporting or not doing
experiments properly.”
The consequence of poor methodology is that many
published papers contain erroneous conclusions, says Mikael
Kubista, a coauthor of the MIQE guidelines and chief executive of the
TATAA Biocenter in Göteborg, Sweden. “The problem is that the
technique itself seems so simple and so easy to do, (but) in real life
you're analyzing biological samples with complexity.”
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- A MIQE Case Study — Effect of RNA Sample
Quality and Reference Gene Stability on Gene Expression Data
Published: December 15, 2011; by Sean Taylor, Bio-Rad Laboratories
Canada, 1329 Meyerside Dr Mississauga, ON, L5T 1C9, and Marguerite
Buchanan and Mark Basik, McGill University, Jewish General Hospital,
Montreal, QC
Published: December 15, 2011
Abstract - Real-time quantitative PCR (qPCR) has become the gold
standard for validating DNA microarray data and is routinely used to
determine gene expression differences between a wide variety of
samples. The exquisite sensitivity of the technology permits the
detection of a single copy of a target gene in a sample which has led
to qPCR now being used in the clinical setting to diagnose infection
and disease states. In an effort to standardize the design of the
associated experiments, the minimum information for publication of
quantitative real-time PCR experiments (MIQE) guidelines were published
in 2009. In this study, we show how qPCR can lead to erroneous
conclusions regarding differences in MCM7 gene expression between
normal and tumor human breast cancer samples if the key steps set out
in the MIQE guidelines are not followed.
- RESULTS: Delivering solutions across an
entire workflow solution
What is RESULTS? RESULTS is a program designed to deliver solutions
across an entire workflow solution -- from
sample collection to data collection. Additionally, it provides
unparalleled access to the brands and technical innovation you need
today.
The analysis performed in your lab every day rely on one thing ... a
result. Whether you are working on complex research, reporting QC data
for product release or isolation of a gene of interest, you rely on
results to make decisions, execute your next move and take you to the
next step of a process or project. At Fisher Scientific, we recognise
the importance of data quality and are committed to helping you achieve
accurate, reliable results every time.
The evolution of scientific technology makes it essential for you to
have access to brands, technical expertise and the latest products to
ensure your success. Our goal is to deliver the right product solution
to solve your research challenges and improve productivity -- allowing
you to focus on what's most important, the science.
To achieve our goal, Fisher Scientific developed RESULTS, a program
designed to deliver solutions across an entire workflow solution --
from sample collection to data collection. Additionally, it provides
unparalleled access to the brands and technical innovation you need
today.
RESULTS applications include Proteomics, Genomics, Cell Biology,
Microbiology and Separation Science.
- Un enseignant-chercheur co-développe
une application iPhone pour la qPCR.
La qPCR, vous connaissez ? Il s’agit d’une méthode d’analyse des
acides nucléiques (ADN et ARN), notamment appliquée dans
le domaine alimentaire pour quantifier des ADN (bactéries, OGM…)
présents dans un aliment et pouvant en affecter sa
qualité, et en médecine, pour détecter des
cellules cancéreuses, des mutations génétiques ou
encore des malformations de fœtus.
Depuis 2009, un référentiel incontournable destiné
aux chercheurs, le MIQE (Minimum Information for Publication of
Quantitative Real-Time PCR Experiments), liste les informations
minimales à insérer dans une publication exposant des
expériences réalisées par qPCR. A la demande de la
société Bio-Rad (leader dans le domaine de la biologie
moléculaire), Afif Abdel Nour, enseignant-chercheur en biologie
moléculaire, et Michael Pfaffl, de la Technical University of
Munich, en ont développé sa version numérique,
interactive, sous forme d’une application pour iPhone et iPad
intitulée MIQE_qPCR.
Le principe est simple : l’application liste, par thématique,
l’ensemble des items du référentiel MIQE à aborder
dans la publication. Le chercheur coche les items à mesure qu’il
avance dans ses écrits, lui permettant de voir l’avancement de
son projet en temps réel. Une bibliographie sur MIQE ainsi que
la possibilité de partager le projet en cours sont des services
complémentaires inclus dans l’application.
- In search of better real-timePCR data
by Richard Kurtz, In Drug Discovery & Development - 1st October 2011
Real-time quantitative PCR (qPCR) has become the industry standard for
the detection and quantification of nucleic acids. However, the lack of
consensus among researchers on how to best perform and interpret qPCR
experiments is a major hurdle for advancing the technology. This
problem is exacerbated when insufficient experimental detail is given
in published work, impeding the ability of others to accurately
evaluate or replicate reported results.
- The StellARray® system supports
adherence to MIQE guidelines
Autumn 2011 by Martina Reiter in Lonza Resource Notes page 12-13
The increasing number of citations of the MIQE guidelines (minimum
information for publication of quantitative real-time PCR experiments,
Bustin et al., 2009) demonstrates a growing emphasis on standardized
experimental practice for qPCR. Lonza’s SYBR-based StellARray® qPCR
array system offers a simple and reliable system for gene expression
analysis that meets the MIQE standards and makes it easier for qPCR
users to compare and publish their results.
- MIQE Guidelines for publishing - Solaris
qPCR Gene Expression Assays
Thermo Fisher Scientific Copyright © 2011
Solaris qPCR Gene Expression Assays are MIQE compliant, in particular
because sequence information for the assay is provided.
The Minimum Information for Publication of Quantitative Real-Time PCR
Experiments (MIQE) guidelines were published in 2009 and aim to
encourage better experimental practice so that published qPCR data
accurately reflects the true biological picture. MIQE is a set of
guidelines that describe the minimum information necessary for
evaluating qPCR experiments.
The MIQE guidelines focus on the reliability of results to help ensure
the accuracy of scientific literature, promote consistency between
laboratories, and increase experimental transparency. The paper
includes a checklist to support the submission of a manuscript to
publishers/journals. By providing all relevant experimental conditions
and assay characteristics, reviewers are better placed to assess the
validity of the protocols used. Full disclosure of all reagents,
sequences, and analysis methods is necessary to enable other
investigators to reproduce results.
MIQE details should be published either in abbreviated form or as an
online supplement.
Checking that inhibition is not affecting RT-qPCR data is one way to
follow the MIQE guidelines. Checking for inhibition reduces the
likelihood of reporting inaccurate or incorrect data and conforms to
MIQE guidelines, and the Solaris RNA Spike Control Kit is a simple and
effective way to achieve this.
- Posts tagged ‘MIQE’
October 2011 by Canadian BioTechnologist 2.0
Are you
using the right reference genes?
Considering Real-Time PCR for gene expression analysis? Have you tested
multiple reference genes or are you just going to run with your
favorite such as 18S? Check out this article on suitable reference gene
selection before you move forward. It could save you lots of grief in
the long run.
Applications
of MIQE to Real Time Quantitative PCR
In this video, Dr. Sean Taylor, Field Applications Specialist, Bio-Rad
Laboratories, demonstrates how sample quality and reference gene
selection effect data analysis and interpretation in real-time
quantitative PCR (qPCR) experiments. The presentation is in accordance
with the previously published MIQE guidelines.
For enhanced viewing, click on the full-screen mode button on the
bottom right hand corner of the video.
A
Practical Approach to MIQE for the Bench Scientist
In a groundbreaking review published in February 2009, Bustin et al
bemoaned the lack of standardization in Quantitative Real-Time PCR
(qPCR) experimentation and data analysis. In their critique the authors
cite the use of diverse reagents, protocols, analysis methods and
reporting formats which has negatively impacted on the acceptance of
qPCR as a robust quantitative molecular tool. The most serious
technical deficiencies include:
* sample storage
* sample preparation
* sample quality
* choice of primers and probes
* inappropriate data and statistical analysis
- Step up to the MIQE
Drug Discovery - Issue 18 - by Richard Kurtz
“Quality data is paramount for the successful development and potential
approval of a drug candidate”
When it comes to real-time PCR in drug discovery, Richard Kurtz
believes that MIQE guidelines will help create a clear path to better
results.
Polymerase chain reaction (PCR) has evolved into a readily automated,
high throughput quantitative technology. Real-time quantitative PCR
(qPCR) has become the industry standard for the detection and
quantification of nucleic acids for multiple applications, and
particularly for the quantification of mRNA expression levels.
However, a lack of consensus among researchers on how to best perform
and interpret qPCR experiments presents a major hurdle for advancement
of the technology. This problem is exacerbated by insufficient
experimental details in published work, which impedes the ability of
others to accurately evaluate or replicate reported results.
- Steps for a Successful qPCR Experiment
October 2011 by IDT
Quantitative PCR (qPCR) is the method of choice for precise
quantification of gene expression. qPCR can utilize a variety of
probe-based methods such as 5′ nuclease dual-labeled probes, molecular
beacons, FRET probes, and Scorpions™ Probes, or use intercalating
fluorescent dyes such as SYBR. 5′ nuclease assays have the advantage of
the specificity that comes with using a sequence-specific, dual-labeled
probe, and is the preferred technique for gene expression analysis.
This article will focus on 5′ nuclease assay design and experimental
setup considerations that will assist in obtaining accurate and
consistent results.
This article
draws upon information published as the MIQE guidelines: Minimum
Information for Publication of quantitative Real Time PCR experiments.
(2009) Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista
M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer
CT. Clin Chem. 55(4):611–622.
- Evolution of Polymerase Chain Reaction -
Seminal Technology Continues to Be a Work in Progress
Feature Articles: October 1st 2011(Vol. 31, No. 17)
by Carl T. Wittwer
Since the discovery of the polymerase chain reaction (PCR) by the
oligonucleotide chemist Kary Mullis in 1983, the method has
revolutionized molecular biology and clinical diagnostics.
Before PCR, DNA amplification required multiple steps, including
cloning into plasmids, insertion into bacteria, bacterial growth,
isolation of plasmid DNA, and separation of inserts from plasmid
vectors.
In contrast, PCR is performed in vitro as a single step, requiring only
two oligonucleotide primers, a polymerase, and temperature cycling of
the DNA template in the presence of deoxyribonucleotides.
Although its spread was initially limited by restrictive patent
policies, the basic method is now off patent and has become a
democratic cornerstone of molecular biology. Thousands of scientists
have contributed to and expanded the methods and applications of PCR,
including quantification of transcripts after reverse transcription and
PCR followed by cycle sequencing.
Anyone can perform PCR with generic reagents and simple laboratory
instruments, amplifying specific DNA segments by 106-to 109-fold for
further study in genetics, oncology, and infectious disease.
- Myth Busted: A
NanoDrop ND-1000 Spectrophotometric reading is insuffiecient to assess
RNA quality
Application Note by Bio-Rad 5893A
- Assuring Reliability of qPCR & RT-PCR
Results - Use of Spectrophotometry on Nucleic Acid Samples Before
Experiment Improves Outcome
20th September 2011, by Andrew Page & Ilsa
Gomez-Curet in GEN
The polymerase chain reaction (PCR) is a valuable tool used in both
research and molecular diagnostic laboratories because of its
specificity, efficiency, fidelity, and relative ease of use.
Quantitative real-time PCR (qPCR) enables sensitive and accurate
quantitative measurement of nucleic acids. Both qPCR and reverse
transcriptase PCR (RT-qPCR) are used across a wide range of
applications such as gene expression, SNP genotyping, copy-number
analysis, pathogen detection, drug target validation, and measurement
of RNA interference (RNAi).
The quality of qPCR and RT-qPCR results can be negatively affected by
many experimental variables. To ensure the validity of assay results,
sample extraction and preparation steps must be closely monitored, and
the starting material must be well characterized before performing RT
and qPCR assays.
Slight differences in pipetting, lack of instrument calibration,
improper choice of reference genes, incorrect quantification, and/or
use of impure nucleic acid templates can generate erroneous, but
believable, results. Therefore, the use of standardized best practices
to ensure reliable and meaningful results is recommended. To address
the need for standardized qPCR practices, the Minimum Information for
Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines
have been developed.
- Welcome to the MIQE website
August 2011 edited by Stephan Bustin
The technical standard of most publications utilising qPCR to quantify
gene expression is either difficult to judge, since very little
information is provided in the "Materials and Methods" sections of most
papers, or is poor, since normalisation procedures are inappropriate,
most commonly using a single, unvalidated reference gene. The
implications of this are disconcerting, since the peer-reviewed
scientific literature forms the bedrock of current knowledge and
provides the starting point for future experiments.
The site is an experimental
one and its aim is to
* assemble information relevant to qPCR
* provide a forum for questions, suggestions and
criticisms about qPCR and MIQE
* review reagents, plastic ware, instruments,
meetings
* initiate discussion about extension of MIQE into
areas not currently covered by the guidelines
* answer questions about experimental protocol, data
analysis and publishing requirements
Over the next few months this web site will be populated with as much
impartial information as possible. Any opinions expressed will be those
of the (identified) contributor(s) and do not necessarily reflect those
of the curator(s) of this web site.
- Applications of MIQE to Real Time
Quantitative PCR
24th of May 2011- in BioPortfolio - Source: American Biotechnolgist
Sean Taylor, Field Applications Specialist, Bio-Rad Laboratories,
demonstrates how sample quality and reference gene selection effect
data analysis and interpretation in real-time quantitative PCR (qPCR)
experiments. The presentation is in accordance with the previously
published MIQE guidelines. For enhanced viewing, click on the
full-screen mode button on the bottom right hand [...] Original
Article: Applications of MIQE to Real Time Quantitative PCR
- Translate
the MIQE guidelines
Since September 2011 we
provide a direct translation of the MIQE guidelines
in CHINESE,
JAPANESE,
KOREAN and RUSSIAN. Please recognize
this is an automatic and robotic based translation service, and
therefore we provide NO guarantee
about the automatic generated content. It
should help the world wide qPCR community to understand the core
content of the MIQE guidelines.
- qPCR and MIQE Seminar Series
Sigma Aldrich Learning Center
As part of our customer education program, we have provided two
recorded seminar series covering the topics of qPCR and MIQE. The
recorded sessions are intended to provide a high level overview of
these subject matters. We have kept the lessons concise so that you can
enjoy a self-paced learning program.
| Seminar
Title |
Presenter |
Recording
Length
(hours : minutes : seconds) |
| Primer
and Probe Design |
Ashley Heath, PhD |
0:06:32 |
| An
Introduction to qPCR Concepts |
Mudassir Mohammed, PhD |
0:09:37 |
| Selecting a
qPCR Basic Detection Chemistry |
Mudassir Mohammed, PhD |
0:12:35 |
| Choosing
a Fluorophore / Quencher Combination |
Anders Bergkvist, PhD |
0:11:30 |
| Chemistries
for More Challenging qPCR Assays |
Mudassir Mohammed, PhD |
0:15:18 |
| MIQE Concepts |
Marina Wiklander, PhD |
0:03:19 |
| Reference
Gene Validation |
Anders Bergkvist, PhD |
0:12:47 |
| Data
Analysis Guidelines |
Anders Bergkvist, PhD |
0:10:42 |
|
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|
| Seminar
Title |
Presenter |
Recording
Length
(hours : minutes : seconds) |
| MIQE: Assay
Design Considerations |
Tania Nolan, PhD |
0:17:37 |
| MIQE: Sample
Derived Inhibitors |
Tania Nolan, PhD |
0:13:04 |
| MIQE: RNA
Quality Considerations |
Tania Nolan, PhD |
0:15:31 |
| MIQE: RNA
Quantity and RT Considerations |
Tania Nolan, PhD |
0:16:38 |
- MIQE (Minimum Information for the
Publication of Quantitative PCR Experiments) has become the gold
standard for assessing the quality and relevance of qPCR-based
publications.
5 August 2011 - Prof Stephen Bustin, Barts and the London School of
Medicine and Dentistry
The real-time polymerase chain reaction uses fluorescent reporter dyes
to combine DNA amplification and detection steps in a single tube
format. The increase in fluorescent signal recorded during the assay is
proportional to the amount of DNA synthesised during each amplification
cycle. Individual reactions are characterised by the cycle fraction at
which fluorescence first rises above a defined background fluorescence, a
parameter previously known as the threshold cycle (Ct) or crossing
point (Cp), now standardised by MIQE as the quantification cycle (Cq).
Consequently, the lower the Cq, the more abundant the initial target.
This correlation permits accurate quantification of target molecules
over a wide dynamic range, while retaining the sensitivity and
specificity of conventional end-point PCR assays. The homogeneous format
eliminates the need for post-amplification manipulation and significantly
reduces hands-on time and the risk of contamination. MIQE abbreviates
real-time PCR to qPCR, with reverse transcription PCR abbreviated to
RT-qPCR.
- Go, Go Gadgets
July 13 2011 by Katia Caporiccio
For researchers working in life science, the MIQE qPCR app for iPhone
and iPad, sponsored by Bio-Rad, provides resources and checklists
needed to ensure MIQE (Minimum Information for Publication of
Quantitative Real-Time qPCR Experiments) compliance for qPCR
experiments. “In this day and age, everybody has his phone with him at
all times,” said Rachel Scott, senior product manager for Gene
Expression at Bio-Rad (Hercules, CA). “The immediacy helps with
capturing the information as it occurs, on the fly.”
- Research gets APP happy
06/23/2011 by Lisa Grauer
These two new iPhone and iPad apps promise to advance your research
more than any summer intern.
Bio-Rad’s new MIQE qPCR app also comes equipped with lab tools
including extensive qPCR reference information and checklists that
allow researchers to ensure MIQE compliance for their qPCR experiments.
“qPCR is a common lab
technique used by most life science researchers today, but not everyone
conducting PCR is using the technique in a way that ensures its correct
interpretation,” said Rachel Scott, Bio-Rad senior product manager. “As
a result of misinterpretation of qPCR data, significant scientific
conclusions have been retracted for inaccuracies.”
Developed by real-time PCR (qPCR) experts Michael W. Pfaffl, professor
of molecular physiology at Technische Universität München,
and Afif Abdel Nour, associate professor of nurigenomics at the
Institut Polytechnique LaSalle Beauvais, the MIQE qPCR app helps
researchers achieve accuracy, transparency, and reproducibility in
their qPCR experiments by allowing them to monitor MIQE compliance via
color-coded checklists and progress bars. The app also provides expert
advice through direct links to MIQE-related publications and email
addresses of qPCR experts.
- Saving lives one iPhone at a time
Monday, July 25th, 2011 by
www.americanbiotechnologist.com
My iPhone is very precious to me. Until I had an iPhone, I wasn’t aware
of how much I was missing. Now that I am an iPhone owner, I don’t know
how I ever lived without one.
There are tons of awesome apps out there. For molecular biologists
there is the NCBI Blast app, the MIQE app and the qPCR app (among
others). There are also a number of cool health apps such as the urine
blood glucose monitor or STD detector app.
Now another cool app has been added to your iPhone’s medical repitoire.
The Melenoma Risk Assessment Tool by Health Discovery Corporation, is
designed to help users learn about melanoma and identify areas on their
skin which may need attention from a physician specializing in the
diagnosis of melanoma.
Using the iPhone camera feature, users can take a picture of their skin
lesions and moles and within seconds receive a risk analysis of their
uploaded picture being a melanoma. Utilizing your iPhone GPS, MelApp
can refer you to a nearby physician specializing in the diagnosis and
treatment of melanoma for proper medical follow up, without the need to
input a zip code or any personal information. These pictures also can
be stored on MelApp and reviewed for changes in the skin lesions
occurring over time.
- The MIQE iPhone App
Monday, July 25th, 2011 by
www.americanbiotechnologist.com
We have written many posts about the MIQE real time
PCR standards that are basic requirements for anyone engaged in real
time PCR experimets. Now there is a new tool for all ipod/iphone users
to add to their arsenal. A MIQE qPCR app!
The MIQE app helps scientitst review scientific work and check their
own project’s MIQE compliance. Plus, the app includes a list of the
most current qPCR news and events and “emergency” contact numbers that
you can call/email should you have any questions about your qPCR
experiments.
The application was developed by Dr. Afif Abdel Nour, Associate
Professor in Nutrigenomics at LaSalle Beauvais, in collaboration with
Dr. Michael Pfaffl and was sponsored by Bio-Rad Laboratories.
- Implementation of MIQE guidelines to the
StellARray system
July 2011by Lonza AG
The increasing number of citations of the MIQE guidelines (minimum
information for publication of quantitative real-time PCR experiments,
(Bustin et al. 2009) demonstrates a growing emphasis on standardized
experimental practice for qPCR.
The SYBR-based StellARray qPCR Array system from Lonza offers a simple
and reliable system for gene expression analysis that meets the MIQE
standards and makes it easier for qPCR users to compare and publish
their results.
download PDF
- 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) |
2010
- IN CHINESE
The MIQE Guidelines - Minimum
Information for
Publication of
Quantitative Real-Time PCR Experiments
Stephen
A.
Bustin 1,
Vladimir Benes 2, Jeremy A. Garson 3,4, Jan Hellemans
5, Jim Huggett 6, Mikael
Kubista 7,8, Reinhold Mueller
9, Tania Nolan
10,
Michael W.
Pfaffl 11, Gregory L. Shipley 12, Jo
Vandesompele 5, and Carl T.
Wittwer
13,14
Overseas
Laboratory Medicine 2010: 3, 1
- The
Importance of Quality Control During qPCR Data Analysis
Barbara D’haene, Ph.D. & Jan Hellemans, Ph.D. Biogazelle &
Ghent University
International Drug
Discovery 2010
Since its introduction in 1993, qPCR has paved its
way
towards one of
the most popular techniques in modern molecular biology [1]. Despite
its apparent simplicity, which makes qPCR such an attractive technology
for many researchers, final results are often compromised due to
unsound experimental design, a lack of quality control, improper data
analysis, or a combination of these. To address the concerns that have
been raised about the quality of published qPCR-based research,
specialists in the qPCR field have introduced the MIQE guidelines for
publication of qPCR-based results [2]. The main purpose of this
initiative is to make qPCRbased research transparent, but the MIQE
guidelines may also serve as a practical framework to obtain
high-quality results. Within the guidelines, quality control at each
step of the qPCR workflow, from experimental design to data analysis,
is brought to the attention as a necessity to ensure trustworthy
results.....
- The Marketing of Science
December 3, 2010
I am a scientist for profit. This means, as you are well aware, I have
to work with marketing people to generate pretty pictures showing
perfect results with any product that we sell. You know those flyers
and brochures and ads in BioTechniques where a tiny picture of a gel or
a qPCR assay with photoshop perfect curves or bands is plopped on the
page next to some meaningless picture and supposed to convince you to
call or go to a website? Those things.
Before working for a company, I would take a look at those pictures but
I never put much stock into them. I mean, of course they're going to
show perfect data. What else will they show? Their kit sucks next to a
competitor? So marketing data never really did sway me much. I looked
at it, but not in any depth. I guess, I expect there to be some attempt
at science in the ad, but it's merely representative data.
My first biotech job wasn't in marketing. The company I worked
for was and still is considered one of the best in the world and I was
so very proud to be a part of that company. When they would introduce a
new product, the product manager would come present all the beautiful
R&D data proving the product works and it was convincing. I would
walk away from those meetings absolutely positive that this was the
best damn invention in the world and we have geniuses in R&D and
how lucky am I to represent such brilliance.
About this first company, I still do believe that they have geniuses in
R&D. However, since leaving, I feel that their employees are
extremely self-obsessed and self-absorbed but I can understand why they
are that way. It is part of the company culture. But
that isn't the point of this article......... => read more
- The Story of MIQE and its Impact for
Future Publications on qPCR
Questions to an Expert in
qPCR, Stephen Bustin (Ph.D.) - The Story of MIQE and its
Impact for Future Publications on qPCR
November 2010
MIQE is a set of guidelines with some essential (59) and some desirable
(28) check points for the documentation that describes the minimum
information necessary for evaluation of quantitative real-time
polymerase chain reaction experiments. Following these guidelines will
encourage better experimental practice, allowing more reliable and
unequivocal interpretation of quantitative PCR results. More details
can be found on Stephen Bustin’s MIQE homepage: http://www.sabustin.org/
- MIQE – Minimum Information for Publication
of Quantitative Real-Time PCR Experiments
suppoted by Sigma-Aldrich
The potential applications for Quantitative Real-Time PCR (qPCR) have
increased exponentially since the first description (Higuchi, 1993).
However, researchers have been frustrated by complications such as
contamination, insufficient amplification, low sensitivity, and
uncertainty about what constitutes a suitable statistical analysis.
Until recently, there has been a lack of consensus about how to deal
with these obstacles.
An international research team, including Dr. Tania Nolan, Sigma's
Global Manager for Applications and Technical Support, published The
MIQE (pronounced Mykee) Guidelines in 2009 to address the challenges of
performing dependable qPCR measurements. By following MIQE, you are
certain to produce more reliable data and will:
* Promote experimental transparency
* Ensure consistency between laboratories
* Maintain the integrity of the scientific literature
Sigma's qPCR services, including primer and probe designs, assay
protocol development, troubleshooting, and data analysis support,
adhere to MIQE, which allows you to publish or bring your product to
market faster and with confidence. =>
read more
Blitzlicht
MIQE-Richtlinien
Qualität und Richtigkeit von
qPCR-Ergebnissen steigern
Laborwelt December 2010
by Michael W. Pfaffl, TUM, Freising-Weihenstephan
Die Anwendung der quantitativen Polymerase-Kettenreaktion (qPCR) oder
die Kombination der qPCR mit der reversen Transkription (RT) ist zu
einem Routinewerkzeug in der modernen mole-kularbiologischen Forschung
und molekularen Diagnostik geworden. Das Expression Profiling
biologischer Proben auf mRNA- und microRNA-Ebene mittels quantitativer
RT-PCR (RT-qPCR) ist von großem Nutzen und liefert wichtige
Ergebnisse in zahlreichen biologischen Disziplinen. Vor allem in der
Routinediagnostik, der universitären und industriellen Forschung
sowie in der funktionellen Genomforschung ist sie unverzichtbar.
Full issue -
Laborwelt - PCR
Spezial - Dezember 2010 |
|
- A
practical approach to RT-qPCR-Publishing data that conform to the MIQE
guidelines.
Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M.
Bio-Rad Laboratories, Inc., Hercules, CA
94547, USA.
in - The
ongoing Evolution of qPCR -
Methods. 2010 Apr;50(4): S1-5. http://evolution.gene-quantification.info
Given the highly dynamic nature of mRNA transcription and the potential
variables introduced in sample handling and in the downstream
processing steps (Garson et al. (2009)), a standardized approach to
each step of the RT-qPCR workflow is critical for reliable and
reproducible results. The MIQE provides this approach with a checklist
that contains 85 parameters to assure quality results that will meet
the acceptance criteria of any journal (Bustin et al. (2009)). In this
paper we demonstrate how to apply the MIQE guidelines
(www.rdml.org/miqe) to establish a solid experimental approach.
- IDT publishes free downloadable qPCR
user
guide
8 December 2010
User guide provides a
MIQE compliant
overview of this essential research technique!
Integrated DNA Technologies has developed an extensive quantitative
real-time polymerase chain reaction (qPCR) user guide, which is
available as a free download.
The manual provides user guidance on the entire qPCR process - from RNA
isolation to data analysis - covering the basics of experimental
set-up, performance and analysis. Specific information on 5’ nuclease
assays, including re-suspensions and qPCR protocols are also supplied,
as well as a troubleshooting section which discusses commonly
encountered issues. The document is written in compliance with MIQE
guidelines: minimum information for publication of quantitative
real-time PCR*. www.idtdna.com
*Bustin et al. The MIQE guidelines:
minimum information for publication of quantitative real-time PCR
experiments. Clin Chem, 2009 55(4): 611-622
- Life Technologies’ TaqMan® Assays QPCR
Guarantee Program Sets New Industry Standard for Customer Service and
Support
by Sam Raha, Vice President and General Manager of Genomic Assays
19 November 2010
Today we are launching Life Technologies’ TaqMan® Assays QPCR
Guarantee Program, which is designed to provide customers with
unparalleled peace of mind by offering to replace any of our more than
7 million pre-designed TaqMan® assays that don’t meet their
expectations (see QPCR video).
The program helps
take some of the risk out of their research by guaranteeing the quality, performance,
content, and results (hence the acronym “QPCR”), across Life Technologies’ line of
pre-designed
TaqMan® Assays. If
for any reason a pre-designed assay does not perform to the level of
our customers’ satisfaction, Life Technologies will first help
troubleshoot the issue and, if unsuccessful, we will replace the assay
or credit their account.
This is Life Technologies’ way of formalizing our
commitment to our customers by standing behind the industry’s
best-performing and most-consistent pre-designed qPCR assays.
TaqMan®
pre-designed assays are used in laboratories around the world among
clinical, pharmaceutical, agricultural and academic researchers.
They serve as powerful tools to rapidly and precisely measure genomic
and proteomic changes in studies that are applied toward disease
research, drug discovery, and agricultural development.
Certain restrictions apply. For details and complete
terms and conditions regarding the TaqMan® Assays QPCR Guarantee,
please visit www.appliedbiosystems.com/taqmanguarantee
- Gene Expression
Assay Performance Guaranteed With the TaqMan® Assays QPCR Guarantee
Program
Real-time or quantitative PCR (qPCR) is one of the most powerful and
sensitive techniques available for gene expression analysis. It is used
for a broad range of applications, including quantification of gene
expression, measuring RNA interference, biomarker discovery, pathogen
detection, and drug target validation. When studying gene expression
with qPCR, scientists usually investigate changes—increases or
decreases—in the quantity of particular gene products or a set of gene
products. Investigations typically evaluate gene response to biological
conditions such as disease states, exposure to pathogens or chemical
compounds, the organ or tissue location, or cell cycle or
differentiation status.
- Publishing
Data That Conform to the MIQE Guidelines
Minimum information for publication of Quantitative Real-Time PCR
Experiments (MIQE) guidelines help researchers design qPCR experiments.
- Are you
MIQE compliant?
2010 by Premier Biosoft International
Since its introduction, real-time PCR has become the main technical
platform for nucleic acid detection in research and development. This
technology has become an invaluable tool for many scientists working in
different disciplines. Especially in the field of molecular
diagnostics, real-time PCR - based assays have gained favor in the
recent past. Although many significant results have been derived from
real time PCR studies, one limitation has been the lack of standards to
perform and interpret these experiments.
The Minimum Information for Publication of Quantitative Real-Time PCR
Experiments (MIQE) guidelines outline the minimum information that
should be included while describing a real time PCR experiment, to
standardize the results, to easily interpret them and to independently
verify them. MIQE guidelines have been written to promote consistency
between laboratories and increase experimental transparency.
Download the MIQE Checklist
Is your experiment MIQE
Compliant?
AlleleID®
and Beacon
Designer™,
our Real Time PCR oligo design software provide everything a researcher
needs to meet MIQE compliance, making submission for publication review
more efficient.
- Making the most of MIQE
BMC Molecular Biology - October 2010
The Editorial Board of BMC Molecular Biology endorse a new set of
essential MIQE-light guidelines for the reporting of quantitative PCR
data: "MIQE precis", and provide guidance for the suitability of pure
reference gene papers to the journal.
- QC Best
Practices for the qPCR Lab
Live Event: Thursday, July 29, 2010 at 2:00 PM EDT
Moderator: Robert Fee, Editor-in-Chief , Bioscience
Technology
Panelist: Manju Sethi, Senior
Product Manager, Thermo Fisher Scientific
|
MIQE
precis: Practical implementation of minimum standard guidelines for
fluorescence-based quantitative real-time PCR experiments
Stephen A Bustin, Jean-Francois Beaulieu, Jim Huggett, Rolf Jaggi,
Frederick SB Kibenge, Pal A Olsvik, Louis C Penning email and Stefan
Toegel BMC Molecular Biology 2010 - Published:
21 September 2010
The conclusions of thousands of peer-reviewed publications rely on data
obtained using fluorescence-based quantitative real-time PCR
technology. However, the inadequate reporting of experimental detail,
combined with the frequent use of flawed protocols is leading to the
publication of papers that may not be technically appropriate. We take
the view that this problem requires the delineation of a more
transparent and comprehensive reporting policy from scientific
journals. This editorial aims to provide practical guidance for the
incorporation of absolute minimum standards encompassing the key assay
parameters for accurate design, documentation and reporting of qPCR
experiments (MIQE precis) and guidance on the publication of pure
'reference gene' articles. |
MIQE
precis: with reference to reference genes
BioMed Central Blog - Sep 21, 2010
Genes that maintain constant expression under a variety of
circumstances are known as ‘reference genes’. They are vital for
researchers who need to quantify gene expression changes in other genes
and need a ‘reference point’ against which to do so. BMC Molecular
Biology, has to date published around 200 reference gene-related papers
from researchers working in such diverse models as peaches, sharks,
barnacles and glioblastoma to name but a few.
However, to be a true reference gene you need to fulfil a certain list
of criteria and the research field is now united in requesting that all
work be performed to the same accuracy and in accordance with
recommended guidelines. The Minimum Information for Publication of
Quantitative Real-Time PCR Experiments (MIQE) guidelines were launched
over a year ago by an international team of researchers. The aim of
these guidelines was to enable the benchmark technology for measuring
gene expression (quantitative PCR [qPCR]) to become standardised when
reported in research papers. The MIQE guidelines advise on good assay
design and appropriate data analyses for nucleic acid detection and
quantification. BioMed Central supports and promotes initiatives aimed
at improving the reporting of biomedical research, and refers
authors to the MIBBI Portal (of which MIQE is part of) for
reporting biological and biomedical research. Whilst some authors have
included MIQE checklists as supplemental files with their work (for
example here), there has been some debate as to the utility and ease in
doing this in all cases.
After working with several Editorial Board Members from BMC Molecular
Biology, we propose that all researchers wishing to publish qPCR work
do so by adhering to our simpler and more abridged 'light' guidelines –
MIQE précis. We also propose that the majority of reference gene
papers are no longer suitable for publication as ‘pure reference gene
papers’, but this information will need to be incorporated as part of a
larger study. Alternatively, authors may publish these more incremental
(but still potentially useful) pure reference gene articles in BMC
Research Notes to contribute to our topical series: “Quantitative Real Time
PCR normalization and optimization” |
- The
Future of qPCR: Best practices, Standardization, and the MIQE Guidelines
September 30, 2010 - 12 noon Eastern, 9 a.m. Pacific, 4 p.m.
GMT
Quantitative polymerase chain reaction (qPCR) has emerged as a powerful
tool in molecular biology laboratories, both in research and in
diagnostic settings. Even as qPCR grows in popularity, it is being
recognized that there are some challenges associated with the
technology, particularly with respect to reproducibility within and
between laboratories. Fortunately, many of these limitations can be
addressed through a standardized set of best practices. Using the
recently published MIQE guidelines as a foundation, our expert panel
will address the best practices of qPCR, with the goal of providing
researchers with more consistent and reliable data.
register
During the
webinar, the panelists will:
- provide an overview of the MIQE guidelines
- address qPCR applications and primary
challenges
- outline best practices and assay design to get
the best out of your qPCR
- describe the essential quality control steps,
including nucleic acid quantification
- answer your questions during the live Q&A
session.
Participants:
- Stephen A.
Bustin, Ph.D.; Queen Mary, University of London; London, UK
- Gregory L.
Shipley, Ph.D.; University of Texas Health Science Center at
Houston; Houston, TX
- Manju R.
Sethi; Thermo Fisher Scientific; Wilmington, DE
MIQE Guidelines
slowly entering "high impact" journals!
Routine lab method's
accuracy called into question
Catherine Shaffer
Nature Medicine Vol 16, page 349 (2010)
download PDF
link
to Nature Medicine
PCR proponents
=>
Stephen Bustin (left) & PCR inventor Kary
Mullis
|
|
|
In 2002, four
years after first sparking public controversy over whether the measles,
mumps and rubella vaccine causes autism, Andrew Wakefield reported a
possible molecular mechanism for the connection. He claimed that a form
of irritable bowel disease, which he called autistic enterocolitis, was
triggered by the measles virus (Molec. Pathol. 55, 84–90, 2002). That
finding, however, was based on a “defective experimental technique,”
Stephen Bustin, a molecular biologist at Barts and the London School of
Medicine and Dentistry, told a US federal court in 2007. The problem:
Wakefield had incorrectly applied the common laboratory protocol known
as quantitative real-time polymerase chain reaction (qPCR) to come to
his conclusions.
Bustin says this faulty lab work is a problem
shared by many researchers around the world who have turned to qPCR to
measure gene expression. Unlike standard PCR, which can only crudely
quantify levels of DNA, the chemistry behind qPCR allows researchers to
assess such levels more precisely by comparing sequences of interest
against a known reference added to the test tube mix as a control.
But the
reference genes used in qPCR can vary between experiments and
laboratories, which can give misleading results or make it difficult to
compare one study to another. As a result of this and other variables
in the technique, a majority of scientific papers involving qPCR
include flawed methods, say a team of leading qPCR experts. Most qPCR
methods, as reported in the literature, are improperly validated and
irreproducible, Bustin claims.
“If you look at
the literature, you find again and again and again the appalling
quality of qPCR protocols,” says Bustin, who this month repeated his
call for the scientific community to adopt the MIQE guidelines (Methods
50, 217–226, 2010). “There's no excuse for anyone either not reporting
or not doing experiments properly.”
The consequence
of poor methodology is that many published papers contain erroneous
conclusions, says Mikael Kubista, a coauthor of the MIQE guidelines and
chief executive of the TATAA Biocenter in Göteborg, Sweden. “The
problem is that the technique itself seems so simple and so easy to do,
[but] in real life you're analyzing biological samples with complexity.”
Wakefield's 2002
study reported the presence of measles virus in the gut, yet the
authors hadn't included a reverse-transcription step to convert the RNA
virus into DNA in some of their qPCR runs, and so they probably
detected a DNA contaminant, according to Bustin's testimony (Eur.
Pharm. Rev. Dig. 1, 11–16, 2008). This error and others like it could
be prevented by following correct methodology, Bustin says. The MIQE
guidelines, for example, call for a detailed description of the
reagents used in the technique, including the enzyme type used for the
RNA reverse transcriptase step. (A follow-up publication that included
the 2002 paper's corresponding author John O'Leary, of Trinity College
Dublin, among others, used the same methods as the original study and
found no link between measles and autism (PLoS One 3, e3140,
2008). (Neither Wakefield nor O'Leary was available for
comment.)
Not all
researchers are convinced that the MIQE guidelines are the perfect
solution. “There's no doubt that there is a need for improved
standardization,” says Helen Fernandes, director of molecular
diagnostics at the University of Medicine and Dentistry of New Jersey
in Newark, who is helping evaluate protocols for the Clinical and
Laboratory Standards Institute, a global organization supporting
consensus lab procedures. But “we have to consider other views or other
guidelines, as well,” she explains.
Many researchers
might be reluctant to adopt the guidelines unless major journals first
change their publication policies. Journal editors, however, are
hesitant to impose new rules without a broader scientific consensus.
“We would be delighted to embrace the [MIQE] guidelines, but we are not
really persuaded that the guidelines are embraced by the community,”
says Juan Carlos López, editor-in-chief of Nature Medicine,
which does not require that authors adhere to MIQE. This view is
reflected in the policies of most leading journals, including Cell,
Science, Nature, PLoS, New England Journal of Medicine and The Lancet,
which do not mention qPCR data in their instructions for authors,
although many have instructions for other common lab techniques such as
DNA microarrays.
One publisher
that has warmed to MIQE is London-based BioMed Central (BMC). Although
adherence to the principles is not explicitly required of authors, BMC
journal editors and reviewers use them to guide disputes over how qPCR
data should be reported. “Where there has been methodological
information lacking or issues raised about the quality of these
particular experiments, it has been extremely useful to quote the MIQE
guidelines,” says BMC's senior scientific editor Scott Edmunds.
However, BMC has no plans make the guidelines compulsory, he adds.
|
- Meeting
Report - Developments in real-time PCR research and molecular
diagnostics
Stephen A Bustin
Expert Review of Molecular Diagnostics
September 2010, Vol. 10, No. 6, Pages 713-715
This meeting was designed to highlight the wide range of new methods,
instruments and applications that underlie the popularity of
quantitative real-time PCR technology in all areas of life science
research, as well as in clinical diagnostics. It provided a fascinating
snapshot of current trends and novel approaches, as well as important
issues concerning assay design, optimization and quality control issues.
- A
Practical Approach to MIQE for the Bench Scientist
In a groundbreaking review published in February 2009, Bustin et al
bemoaned the lack of standardization in Quantitative Real-Time PCR
(qPCR) experimentation and data analysis. In their critique the authors
cite the use of diverse reagents, protocols, analysis methods and
reporting formats which has negatively impacted on the acceptance of
qPCR as a robust quantitative molecular tool.
- Illumina ecoqpcr
Software
The Eco Real-Time PCR software interfaceEvery Eco system includes a
Netbook computer pre-installed with flexible, easy-to-use software that
integrates user control, real-time data collection, and advanced data
analysis. The software conforms to MIQE (Minimum Information for
Publication of Quantitative Real-Time PCR Experiments) guidelines,
making data analysis and submission for publication review more
efficient.
-
paper MIQE guidelines
-
MIQE guideline checklist
Eco software uses a unique icon-driven user interface to simplify
experimental design and setup. Pre-set defaults for plate setup and
thermal profile are provided for the most commonly used experimental
protocols. Temperature and time for each protocol step can easily be
changed by click-and-drag action with the mouse. Experiment templates
can be customized and saved for future use. All qPCR chemistries
and all standard Real-Time PCR applications are supported, with
High Resolution Melt (HRM) analysis as a standard option.
Efforts to
standardize qPCR data meets mixed reviews
05/25/2010
Uduak Grace Thomas
BioTechniques
One year ago, an
international team of researchers proposed guidelines
for the publication of real-time PCR experiments. Since then, there has
been mixed response from the scientific community. Uduak Thomas
investigates the reasons behind the resistance.
|
|
- qPCR's
Big Bag of Tricks - June 2010 by Tracy Vence
Assuring quality
But as qPCR has been gaining attention for its applications that have
identified new targets, it hasn't been kept properly in check, says
Stephen Bustin, a professor of molecular science at the Queen Mary
University of London. "This attention is accompanied by a reluctance to
question the reliability and relevance of the qPCR data," Bustin says.
"Unlike many diagnostic assays it is threatening to replace," he
continues, "qPCR is not a mature technology — there are serious
disagreements on how best to perform the assay, how to obtain copy
numbers or relative quantification data from raw quantification cycles,
and whether linear regression or non-linear regression algorithms are
most suitable for data analysis."
In a 2009 Clinical Chemistry paper, Bustin and other PCR experts
suggested the minimum information for publication of quantitative
real-time experiments, or MIQE. Because Bustin et al. note that "full
disclosure of all reagents, sequences, and analysis methods is
necessary to enable other investigators to reproduce results," they
argue that MIQE details should accompany every peer-reviewed qPCR
paper, whether in an abbreviated form or as a supplement.
Bustin predicts that there will be "continued publication of
contradictory results, persistence of uncertainty, and, consequently,
lack of confidence by clinicians in PCR data as the basis for their
diagnostic and prognostic decision-making" until there is a
community-wide agreement on how to best standardize the technology.
Ghent's D'haene says a "big challenge remains [in the] adherence of
qPCR-based scientific articles to the recently published MIQE
guidelines." In abiding by standardized practices, she says, studies
will become "much more transparent, reproducibly in other labs, and
simply lead to higher-quality and trustworthy conclusions."
- The
MIQE Guidelines Uncloaked - Speaker:
Greg Shipley
8 June 2010
The MIQE (Minimum Information for Publication of Quantitative Real-Time
PCR Experiments) guidelines have been presented to serve as a practical
guide for authors when publishing experimental data based on real-time
qPCR. Each item is presented in tabular form as a checklist within the
MIQE manuscript. However, this format has left little room for
explanation of precisely what is expected from the items listed and no
information on how one might go about assimilating the information
requested. This presentation presents an expanded explanation of the
guideline items with commentary on how those requirements might be met
prior to publication.
- Do
Your RT-qPCRs Make The Grade? - Tech Tip
by Suzanne Kennedy
Real-time PCR is a technique that is now commonly employed in almost
all molecular biology laboratories to quickly answer very specific
questions. Northern and Southern blotting are now a thing of the past.
No longer do we wait days to know whether a gene is expressed. We can
have the answer in 45 minutes!
But with the widespread use of such a wonderful and sensitive
technology comes differences in how results are reported in the
literature. There are also differences between reviewers reading these
papers and their understanding of the essential information required to
judge the accuracy of the reported data.
To overcome this increasing problem of lack of consistency in
publications, a panel of real-time PCR experts published a set of
guidelines containing what they consider the minimal information
required when reporting qPCR results. That paper called The MIQE
Guidelines: Minimum Information for Publication of Quantitative
Real-Time PCR Experiments, was published February 2009 in the Journal
of Clinical Chemistry.
- Comply
with MIQE guidelines for qPCR
- Agilent 2100 Bioanalyzer assessment of RNA integrity
by Ruediger Salowsky - Agilent Product Manager Bioanalyzer - RNA/DNA
Solutions
Quantitative real-time polymerase chain reaction (qPCR) and microarray
analysis have become essential for elucidating variations in gene
expression. While guidelines that define the minimum information
required for interpretation of microarray data have been available
since 2001,[1] similar specifications for qPCR experiments have been
developed only recently. In early 2009, a consortium of leading
scientists who use qPCR, established specifications for the minimum
information that you must report for a qPCR experiment that you wish to
publish. These are the MIQE guidelines (for minimum information for
publication of quantitative real-time PCR experiments). This article
describes how the Agilent 2100 Bioanalyzer helps you meet these
requirements.
- The MIQE Guidelines -
Minimum Information for Publication of Quantitative Real-Time PCR
Experiments
initiative by PrimerDesign
During the past decade, several high-profile cases of faulty research
have been linked to inconsistent real-time PCR techniques and
experiments. In April 2009, Stephen Bustin, a molecular science
professor at the school of medicine and dentistry at Queen Mary
University of London and an international team of nine scientists,
joined forces and developed a set of guidelines for the publishing qPCR
results. The resulting 'MIQE guidelines' outline the minimum
information required to publish quantitative real-time PCR data with
scientific integrity.
PrimerDesign is cited in the MIQE guidelines because we share the same
philosophy on primer sequences as the authors. We have always provided
the primer sequences with our custom designed assays as we believe that
this is crucial to perform research with integrity.
We strive to make all of our products compliant with the MIQE
guidelines and will always be on hand to guide and advise you in
producing real-time PCR data of the highest quality.
- High-Impact
Journals, Large Vendors Contributing to Lack of Quality Control for
qPCR Data Publication
June 03, 2010 - By Ben Butkus
GÖTEBORG, Sweden – Quality control for publication of quantitative
PCR data is still severely lacking – and, in the case of high-profile
scientific journals such as Nature and Science, it is "absolutely
appalling and scandalous" – according to Stephen Bustin, one of the
scientists leading the drive to adopt a set of standards intended to
guide high-quality and reproducible qPCR experiments within the
field..............................
- Following
MIQE Recommendations - EMBL
Heidelberg, Germany
Monday 5 July - Friday 9 July 2010
Since the early descriptions of the use of quantitative Real Time PCR,
the technique has been adopted in almost every aspect of life science
research and is increasingly used for clinical analysis. Over time
protocols and strategies have been tried and tested, amended and
developed such that there are currently several different approaches.
Protocol variations are evident at each step of the RT-qPCR process,
from sample acquisition to data analysis (e.g. sample QC, experimental
design, assay design and validation, normalisation, biostatistical
interpretation, reporting, etc). It is now apparent that these
adaptations may result in differences in the final biological
conclusion of the study.
This workshop is based upon the MIQE guidelines. Each step of the
RT-qPCR process will be discussed and protocol variations illustrated
practically. The student will be instructed in best practice and
acceptable alternative strategies.
- How
to design your qPCR experiment so that it conforms to the MIQE
guidelines
There have recently been some high profile retractions of scientific
papers that have reported data which turns out to be artifacts of
poorly designed experiments. The MIQE guidelines is a daunting
list of 85 items that need to be addressed before a reviewer will
accept your qPCR results as valid.
In the PDF below, the essential elements of sample preparation and
experimental design are outlined as a practical guide to meeting the
MIQE guidelines. If you use the approach followed in this guide,
you will save countless hours of effort trying to figure everything out
on your own. Sean Taylor, Michael Wakem, and Greg Dijkman
have contributed their many years of qPCR experience, and when you
follow this guide, your gene expression experiment will meet the MIQE
guidelines.
Onsite hands-on training for qPCR gene expression studies is
available. Please use the contact form to ask for a quote, and
make sure you include your location (city).
This is a reprint of the article
published in Methods: April 2010 - Practical Guide to MIQE guidelines
Now is a great time to consider an instrument like the Experion to
validate the quality of your RNA. If you do not already have a
Bioanalyzer or an Experion, you will need to run your RNA samples out
on a gel.
- Professor
calls for urgent change in research methods after Dr Andrew Wakefield
is struck off
Monday 24 May 2010
Disgraced Dr Andrew Wakefield used research methods which were flawed -
but which remain commonplace in the scientific world, according to a
professor who gave evidence against him.
Professor Stephen Bustin, based at Queen Mary University London, was
one of the research scientists who gave evidence against Dr Wakefield
in 2007, and about the quality of the science he used to prove his
now-discredited theory about the MMR vaccine.
- Nucleic
Acid Electrophoresis
Monday April 12, 2010 by Catherine Shaffer
Don't have time to pour your own agarose gel, make your own buffers,
and wait more than an hour for the results? You're in luck, because
nucleic acid electrophoresis is getting easier every day. Using precast
gels, all-in-one kits, and even automation, you can make a
time-consuming, tedious task as simple as microwaving a bag of
popcorn—and get better results than your grandfather did when he was
pouring his own gels back in 1990. And although agarose and
polyacrylamide gel electrophoresis are reliable, rock-solid techniques
that have been in use for decades, there are still some surprising
innovations to be made by thinking outside the gel box.
-
March/April
2010
Online
version in American Biotechnology Laboratory
-
A
Practical Guide to Publishing RT-qPCR
Data That Conform to the MIQE guidelines
In an effort to
assist the scientific com-
munity in producing consistent, high- quality data from qPCR
experiments, the minimum information for publication of
quantitative real-time PCR experiments (MIQE)
guidelines has been recently published.
- Documenting
Real-Time PCR - by Catherine Shaffer, Contributing Editor
Drug Discovery & Development - April 01, 2010
In February 2009, twelve internationally recognized experts published a
long-awaited set of guidelines for real time PCR experiments after more
than a decade of public discussion of how to standardize the method and
its reporting. These, guidelines called Minimum Information for
Publication of Quantitative Real-Time PCR Experiments (MIQE),1 can be
found online at www.rdml.org/miqe.php.
The spirit of the guidelines is to standardize and streamline the
real-time PCR workflow from the early planning stage through
publication. Many real-time PCR experiments currently suffer from a
lack of standardization and detail in publication. Some of them are
flawed by poor experimental design. Although the guidelines are barely
a year old, awareness and use of MIQE is not spreading as quickly as
many of the thought leaders had hoped. Instrument and reagent vendors
are helping out by providing MIQE compliant products and MIQE training
resources for customers.
- Step
up to the MIQE by Richard
Kurtz
Tuesday, March 30, 2010
Over the years, polymerase chain reaction (PCR) has
evolved into a
readily automated, high throughput quantitative technology. Real-time
quantitative PCR (qPCR) has become the industry standard for the
detection and quantification of nucleic acids for multiple application,
including quantification of RNA levels. But a lack of consensus among
researchers on how to best perform and interpret qPCR experiments
presents a major hurdle for advancement of the technology. This problem
is exacerbated by insufficient experimental detail in published work,
which impedes the ability of others to accurately evaluate or replicate
reported results......
- Real-time PCR
on SciTopics
UPDATE on 21 January 2010 by Prof Stephen
Bustin
Category: Biochemistry, Genetics and Molecular Biology
Guidelines for minimum information required for publication of qPCR
data have now been published in Clinical Chemistry
- qPCR Assay Quality
Assessment on SciTopics
UPDATE on 21 January 2010 by Prof Stephen
Bustin
Category: Biochemistry, Genetics and Molecular Biology
Guidelines for minimum information required for publication of qPCR
data have now been published in Clinical Chemistry
2009
- REVIEW
of the MIQE publication by UHN Mircoarray Centre, Toronto, Canada
Summary of: Bustin SA, et al. The MIQE Guidelines: Minimum Information
for Publication of Quantitative Real-Time PCR
Experiments. Clinical Chemistry 2009, 55(4):611-622
- Standardization of qPCR Data Reporting
by Kirsteen H. Maclean Ph.D.
Since its discovery by Kary Mullis and colleagues in 1983, the use of
the polymerase chain reaction (PCR) has been a mainstay of scientific
research and discovery [1]. Indeed in discussing its inaugural
“Molecule of the Year” in 1989, the journal Science provided a concise
explanation as to the simplicity of the PCR process:
"The starting material
for PCR, the 'target sequence,' is a gene or segment of DNA. In a
matter of hours, this target sequence can be amplified a million fold.
The complementary strands of a double-stranded molecule of DNA are
separated by heating. Two small pieces of synthetic DNA, each
complementing a specific sequence at one end of the target sequence,
serve as primers. Each primer binds to its complementary sequence.
Polymerases start at each primer and copy the sequence of that strand.
Within a short time, exact replicas of the target sequence have been
produced. In subsequent cycles, double-stranded molecules of both the
original DNA and the copies are separated; primers bind again to
complementary sequences and the polymerase replicates them. At the end
of many cycles, the pool is greatly enriched in the small pieces of DNA
that have the target sequences, and this amplified genetic information
is then available for further analysis."
- Step
up to the MIQE by Richard Kurtz
When it comes to real-time PCR in drug discovery, Richard Kurtz
believes that MIQE guidelines will help create a clear path to better
results. Polymerase chain reaction (PCR) has evolved into a readily
automated, high throughput quantitative technology. Real-time
quantitative PCR (qPCR) has become the industry standard for the
detection and quantification of nucleic acids for multiple
applications, and particularly for the quantification of mRNA
expression levels. However, a lack of consensus among researchers on
how to best perform and interpret qPCR experiments presents a major
hurdle for advancement of the technology. This problem is exacerbated
by insufficient experimental details in published work, which impedes
the ability of others to accurately evaluate or replicate reported
results.........
- Le linee guida
MIQE - The MIQE guidelines - talk by Paolo Scaruffi
- MIQE
Guidelines 'Slowly Filtering Through' PCR Community Despite Lack of
Journal Enforcement
December 31, 2009
Although the guidelines are beginning to catch on among researchers and
vendors, they appear to have made little or no impact on the quality of
the published literature over the last year.
- Videos
explaining MIQE guidelines
November
11, 2009
Browsing through You Tube just now, I found these
videos illustrating
the concepts of the MIQE (Minimum Information for Publication of
Quantitative Real-Time PCR Experiments) guidelines. These focus on how
to apply the guidelines to design a solid experimental approach for
RT-qPCR. There are four videos in total. The sound is a bit
“fuzzy,” but the content is a fairly nice overview of MIQE.
- Helixis Tutorial:
MIQE guidelines: a bench perspective on use and benefits
Description: "MIQE guidelines from a scientist perspective and
discussion on their use and benefits when performing Real-Time PCR
experiments. " Total Running Time: 9:52 (posted
10/29/2009)
Direct YouTube link => http://www.youtube.com/watch?v=zm9QoIpOzkM
- MIQE
checklist
http://www.helixis.com/support/usefultools/MIQE_Checklist.pdf
Description: To help you follow the latest MIQE
guidelines, Helixis has formatted this useful checklist to keep handy
at your bench or desk when designing your Real-Time PCR experiments or
drafting your next paper.
- New Standards for qPCR and RT-qPCR
September 3, 2009 by Isobel
Arguably, no technique has had greater impact on the progress of
biomedical research in recent years than quantitative real-time PCR. It
has accelerated the pace of research and opened up exciting
possibilities for detection and treatment of disease. The widepread
adoption of qPCR as a standard technique is evident even in the most
cursory literature search; the term “real-time PCR” returns over 14,000
papers published in 2009 alone. However, many scientists are concerned
about the lack of standardization of qPCR experiments.
Quality assessment is a
big fat elephant sitting in the
room: everyone knows what needs to be done, but most researchers do not
follow basic quality control guidelines. This serves to undermine the
integrity of the scientific literature to such an extent, that a high
proportion of publications are reporting technical or analytic
artifacts”. Prof. Stephen Bustin, April 2009 SciTopics article.
The publication of a comprehensive set of guidelines
for quantitative, real-time PCR highlights a need for greater
consistency and standardization in reporting the results of qPCR and
RT-qPCR analyses. The MIQE (Minimum Information for Quantitative
Real-Time PCR) guidelines, published in the April 2009 edition of Clinical Chemistry,
seek to create global consensus on how to best perform qPCR experiments
and how to report qPCR results.
The paper, published by an international group of
scientists from
institutions throughout Europe and the United States, seeks to address
issues from basic nomenclature (Cq, vs Ct, Cp,
or TOP) to quality control of nucleic acids to oligo design and assay
normalization. Some of the topics covered in detail are:
- Sample processing
- DNA and RNA quality and integrity
- Appropriate controls
- Comprehensive reporting of reagents, plasticware
and protocols
- Inclusion of oligo sequences and accession
numbers of target genes
- Publication of primer sequences
- Considerations of target secondary structure and
specificity of oligos for target
- Inclusion of information on the validity of the
reference genes for the sample type used
- Comprehensive reporting of data analysis methods
The stated aim of the
guidelines is to support the integrity of the
scientific literature, promote consistency between labs, and increase
experimental transparency. The MIQE checklist contains a list of
mandatory and required elements that at first glance is somewhat
daunting. However, the required elements are clearly there to help
consistency, transparency, accuracy and reproducibility, and many of
them, such as reporting the accession number of the target gene, and
accurately identifying the reagents and protocols used, do not appear
to be cumbersome. Most address the need for commonsense controls,
accurate descriptions of sample handling processes, and consistency in
nomenclature and normalization of results.
A related GEN
article, published in August states: "Adoption of the mandatory
guidelines as a first strategy
assures that key parameters affecting data quality are being addressed
immediately and will have a swift impact on confidence levels in the
data and the conclusions drawn from it”
The authors are actively
seeking feedback from the research
community on these guidelines and consider them to be a constantly
evolving document that is very much a work in progress.
- Do
Your RT-qPCRs Make The Grade?
26th July 2009 - Real-time PCR is a technique that is now commonly
employed in almost all molecular biology laboratories to quickly answer
very specific questions. Northern and Southern blotting are now a thing
of the past. No longer do we wait days to know whether a gene is
expressed. We can have the answer in 45 minutes!
But with the widespread use of such a wonderful and sensitive
technology comes differences in how results are reported in the
literature. There are also differences between reviewers reading these
papers and their understanding of the essential information required to
judge the accuracy of the reported data.
To overcome this increasing problem of lack of consistency in
publications, a panel of real-time PCR experts published a set of
guidelines containing what they consider the minimal information
required when reporting qPCR results. That paper called The
MIQE Guidelines: Minimum
Information for Publication of Quantitative Real-Time PCR Experiments,
was published February 2009 in the Journal of Clinical Chemistry.
This is not only a great resource for authors, but it also essentially
a troubleshooting guide as well. If you don’t have an answer to each of
the item on the checklist, then maybe you are missing an essential
piece of information in your experiment.
- Publishing
Data That Conform to the MIQE Guidelines
Minimum information for publication of Quantitative Real-Time PCR
Experiments (MIQE) guidelines help researchers design qPCR experiments.
Real-time quantitative polymerase chain reaction (qPCR) is a definitive
technique for quantifying differences in gene expression levels between
samples. However, a lack of consistency in experimental design and
reporting combined with inadequate guidelines to review submitted
articles with qPCR data greatly increases the potential of reporting
statistically insignificant and conflicting results.1 The publication2
and retraction3 of a Science “Breakthrough of the Year 2005” article
underlines the issue.
- MIQE
Guidelines 'Slowly Filtering Through' PCR Community Despite Lack of
Journal Enforcement
by Bernadette Toner Genome Web
Guidelines proposed in early 2009 to help standardize how qPCR results
are reported are "slowly filtering through" the research community, but
much work still needs to be done to improve the quality of published
qPCR studies, according to one of the authors of the standard.
- Are
your qPCR
experiments compliant with MIQE?
The MIQE guidelines establish specifications for the minimum
information that must be reported for a qPCR experiment in order to
ensure its relevance, accuracy, correct interpretation and
repeatability. Comply
with MIQE guidelines !
Learn why Prof.
Kubista from the TATAA Biocenter uses the Agilent 2100 Bioanalyzer for
RNA quality control => Start
webinar
- Feature
Article - PCR Technology Review:
Standardization of qPCR and RT-qPCR -
New Guidelines Seek to Promote Accurate Interpretation of Data and
Reliable Results
by Stephen A. Bustin, Jo Vandesompele, Michael
W. Pfaffl
=> download
PDF
The perceived ease of use of real-time quantitative PCR (qPCR) and
reverse transcription PCR (RT-qPCR) technology has revolutionized life
science research. Its effectiveness at amplification and quantification
of low levels of nucleic acids has driven the emergence of numerous
applications, including cellular mRNA and miRNA quantification,
biomarker discovery and validation, microbial quantification, cancer
risk assessment, gene dosage determination, and detection of extremely
low copy targets for forensic investigations. This, in turn, has
resulted in an abundance of publications utilizing qPCR data obtained
with diverse reagents, protocols, analysis methods, and reporting
formats. Unfortunately, few papers report in detail how these results
were obtained. This lack of clarity and transparency has led to concern
in the research community over the reliability of qPCR data
interpretation and the real danger of the scientific literature being
corrupted with publications reporting erroneous and conflicting
results. This has already occurred in some cases, resulting, for
example, in retraction of a Science “Breakthrough of the Year 2005”
report. Now that qPCR has come of age, standardization is needed to
ensure its validity, prompting the recent formulation of guidelines to
increase experimental transparency, promote consistency between
laboratories, and therefore, help assure the publication of valid
conclusions.
- A practical approach to
RT-qPCR - Publishing data that conforms to the MIQE guidelines
(Bio-Rad amplification tech note 5859)
by Sean Taylor, et al., Bio-Rad
Laboratories, Hercules, CA
- MIQE Guidelines -
RNA Qualitätskontrolle in der Genexpressionsanalytik – ein
Meilenstein auf dem Weg zum Erfolg (in German) by Christiane Becker,
Irmgard Riedmaier, and Michael W. Pfaffl
Abstrakt (D) - Die Qualität des Probenmaterials, also der
Gesamt-RNA, hat einen markanten Einfluss auf die Richtigkeit der
quantitativen RT-PCR. Die Überprüfung der RNA Qualität
vor einer Expressionsmessung ist unabdingbar, um verlässliche
RT-qPCR Expressionsergebnisse zu erhalten.
Abstract (E) - The integrity of total RNA has a distinct influence on
the accuracy of RT-qPCR. Quality assessment is an essential step for
the evaluation of reliable results in gene expression analysis.
- Press
release
Standardization of qPCR and RT-qPCR -
New Guidelines Seek to Promote Accurate Interpretation of Data and
Reliable Results
http://pressemitteilung.ws/node/166061
- International Scientists
Secure Quality in Molecular Diagnostics
SALT LAKE CITY, March 31, 2009
- ARUP Laboratories and the American Association for Clinical Chemistry (AACC) announced today that a consensus
guideline for a key laboratory method called qPCR (or quantitative
polymerase chain reaction) was published by a group of international
scientists representing the medical and research fields.
- Consensus Guideline Reached For Quantitative
Polymerase Chain Reaction
Press release by TATAA Biocenter
Gothenburg, March 31, 2009 - TATAA BIOCENTER and
the American Association for Clinical Chemistry (AACC), announced
today that a consensus guideline for a key laboratory method called
qPCR (or quantitative polymerase chain reaction) was published by a
group of international scientists representing the medical and research
fields.
- Internationale Wissenschaftler sorgen für
Qualitätssicherung in der Molekulardiagnostik
Salt Lake City (ots/PRNewswire) - - Einigung über
Konsensus-Richtlinie bezüglich der quantitativen
Polymerase-Kettenreaktion erzielt ARUP Laboratories und die American
Association for Clinical Chemistry (AACC) gaben heute bekannt, dass
eine Konsensus-Richtlinie für die wichtige, qPCR (quantitative
Polymerase-Kettenreaktion) genannte Labormethode veröffentlicht
worden sei. Verantwortlich für die Veröffentlichung war eine
Gruppe internationaler Wissenschaftler als Vertreter der Gebiete
Medizin und Forschung.
- Real-timePCR data markup language
The aim of MIQE, coordinated by a group of research-active scientists
and coordinated under the umbrella of MIBBI (Minimum Information for
Biological and Biomedical Investigations http://www.mibbi.org)
is to provide authors, reviewers and editors specifications for the
minimum information that must be reported for a qPCR experiment in
order to ensure its relevance, accuracy, correct interpretation and
repeatability. A checklist, which should be submitted along with the
paper, is available for authors in preparing a manuscript employing
qPCR.
http://www.rdml.org/guidelines.php
- Letter of the MIQE authors
Letter to leading journals recommending the use of MIQE for quality
control of qPCR experiments. Download
letter PDF
- IBT of the Academy of
Sciences of the Czech Republic
PRAGUE, April 1, 2009
- Institute of Biotechnology of the Academy of Sciences of the
Czech Republic, v.v.i. (IBT) and the American Association for Clinical
Chemistry (AACC), announced today that a consensus guideline for a key
laboratory method called qPCR (or quantitative polymerase chain
reaction) was published by a group of international scientists
representing the medical and research fields.
- qPCR Grows Up by genome web
Bustin is now at the
forefront of a movement to get researchers to follow a set of
guidelines, the minimum information for publication of quantitative
real-time PCR experiments, or MIQE, that were published online at
Clinical Chemistry in February.
"In my talks, I always refer to the cowboy stage of
qPCR. For quite a while everything went," Bustin says. In particular,
he casts a critical eye on how people have been normalizing their gene
expression data. In northern blot and standard PCR experiments that
didn't give quantitative data, people often used a single reference
gene. "People just moved that approach to qPCR without thinking about
what they were doing," Bustin says. "Are these reference genes really
invariant or are they changing with treatment?"
- qPCR Assay Quality
assessment on SciTopics
8 April 2009 by Prof Stephen
Bustin; Category: Biochemistry, Genetics and Molecular Biology
Guidelines for minimum information required for publication of qPCR
data have now been published in Clinical Chemistry
qPCR quality
assessment relates mainly to the reverse transcription -qPCR (RT-qPCR)
variant of the technology. This is widely used to measure pathogen as
well as cellular RNA copy numbers; the former, given appropriate
standard operating procedures and technical expertise, is fairly
straightforward. The latter can be highly problematic. For both types
of assay, however, RNA quality is a major consideraton.
Quality assessment is a big fat elephant sitting in the room: everyone
knows what needs to be done, but most researchers do not follow basic
quality control guidelines. This serves to undermine the integrity of
the scientific literature to such an extent, that a high proportion of
publications are reporting technical or analytic artifacts.
Incredibly, many researchers are not bothered by this; indeed some have
been heard to remark that they can't be bothered assessing RNA quality,
worrying about reverse transcription or determining what normalisdation
strategy to follow. However, efforts are underway to establish a
checklist for journal editors and reviewers, with the aim of
introducing a minumum standard of assay reporting.
- Quest Agrees to Pay Fine for Misbranding Tests
First-ever consensus guidelines
on quantitative PCR aim to improve the quality and transparency of
studies involving qPCR (Clin Chem, 2009; 55: 611-622). The Minimum
Information for Publication of Quantitative Real-Time PCR Experiments
(MIQE) guidelines outline the minimum information necessary to evaluate
qPCR studies, including all relevant experimental conditions and assay
characteristics, and full disclosure of all reagents, sequences, and
analysis methods. The guidelines include an 85-item checklist of
desirable and essential steps to be followed when using qPCR and
information to be divulged from experiments involving qPCR. The purpose
of the guideline is to encourage better experimental practice, so as to
enable more reliable and unequivocal interpretation of qPCR results.
Use of qPCR has
proliferated, yet studies “invariably use diverse reagents, protocols,
analysis methods, and reporting methods,” the authors wrote. “This
remarkable lack of consensus on how best to perform qPCR experiments
has the adverse consequence of perpetuating a string of serious
shortcomings that encumber its status as an independent yardstick.” If
researchers follow the guidelines, they should be able to design and
report qPCR experiments with greater inherent value, and fellow
researchers, editors, and laboratorians should be able to evaluate the
technical quality of the published data against an established standard.
- Advancing DNA research
safely and securely
27 May 2009; Dr Jeremy
Garson& Dr Jim Huggett - Dr Jeremy Garson (UCL Centre for Virology)
and Dr Jim Huggett (UCL Centre for Infectious Diseases and
International Health) have been at the heart of developing a new set of
guidelines on the way scientists the world over use qPCR – a technology
crucial to forensic analysis and diagnosing diseases. Below Dr Huggett
explains how and why they went about it.
What do you hope to
achieve with the guidelines?
By
developing the MIQE guidelines, we aim to
enable researchers to perform high-quality qPCR that allows their
experiments to be easily understood and repeated by workers in
laboratories anywhere in the world. For science to advance swiftly and
securely it is essential that the results of experiments can be
independently reproduced.
- Data that Meets the MIQE Guidelines
Canadian BioTechnologist 2.0 on 27 May 2009 - Key Steps to Generating
High Quality Real-Time PCR (RT-qPCR) Data that Meets the MIQE
Guidelines Speaker: Sean Taylor, Ph.D., Bio-Rad Laboratories PDF slide deck.
- GLOSSARY
OF REAL-TIME PCR TERMS by M.Tevfik Dorak
MIQE - An initiative by the International Real-time PCR Data
Markup Language (RDML) Consortium to generate a structured and
universal data standard for exchanging quantitative real-time PCR
experiment data. This effort resulted in standard guidelines for
reporting qPCR data (publication checklist: XLS,
PDF)
2008
It
is crucial that data acquisition, analysis and reporting become more
transparent to allow reinterpretation and to guarantee compliance with
quality standards. Therefore, following the example of the microarray
community and their MIAME (Minimum Information About a Microarray
Experiment) guidelines, we propose guidelines specifying the minimal
information about qPCR experiments. A RDML guidelines compliant RDML
file should contain all measured data as well as information about the
samples and targets being analyzed.
In
addition, data must be linked to samples and targets in an unequivocal
way. Due to the complexity and diversity of experiments in which qPCR
is utilized, the scope of the RDML guidelines is limited to the
technology itself, which means that these guidelines can easily be
integrated into other minimum information guidelines that focus on the
wider experimental context. To coordinate this effort, the RDML
consortium recently joined the MIBBI project (Minimum Information for
Biological and Biomedical Investigations). The minimum information
guidelines have been kept minimal to facilitate the creation of a
compliant RDML files that make the least demand on researchers’ time,
while requiring sufficient information for other researchers to
interpret and reanalyze the data contained within an RDML guidelines
compliant RDML file.
2006
- How
Reliable is Your qPCR Data?
Drug Discovery & Development - March 01, 2006
These excerpts from a recent Webcast on quantitative polymerase chain
reaction for gene expression analysis involve experts from industry and
academia discussing their experiences with, and data gained from, the
method.
Senior Editor Patrick McGee recently hosted a Webcast entitled "How
reliable is your qPCR data?" Quantitative PCR is a powerful and
sensitive technology for the quantification and validation of genetic
data. Despite the power of qPCR, however, a number of key
considerations need to be addressed, from sample preparation through
data analysis. Although the topic of the day was overcoming the
challenges of qPCR, from pre-assay through data analysis, panelists
limited their comments to gene expression analysis. The full version of
the Webcast is available for viewing at www.dddmag.com/qpcr.
The panel of experts who joined McGee for the Webcast included Stephen
Bustin, PhD, professor of Molecular Science at Barts and the London
Queen Mary's School of Medicine and Dentistry at the University of
London. His research group focuses on molecular oncology and has spent
the last eight years working on applying molecular techniques such as
qRT-PCR to the biology of colorectal cancer. Mark Anderson, PhD,
research and development scientist, Invitrogen Corp., has extensive
experience in analyzing and developing PCR technology and he discussed
qPCR assay design and troubleshooting. Maurice Exner, PhD, research and
development manager in infectious diseases at Quest Diagnostics, is
responsible for directing research efforts to develop new clinical
diagnostic assays for infectious diseases. These assays primarily use
automated nucleic acid extraction methods coupled with various nucleic
acid amplification techniques, particularly qPCR.
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