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MIQE Guidelines are entering "high impact" journals!
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LIFE SCIENCE TECHNOLOGIES - qPCR Innovations and Blueprints
Science, 7. October 2011 by Chris Tachibana
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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.
Routine lab method's accuracy called into question
Nature Medicine Vol 16, page 349 (2010) by Catherine Shaffer in
Nature Medicine
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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, 8490, 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, 611622, 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, 217226, 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
G?teborg, 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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RefGenes - a unique tool to find suitable reference genes
http://www.***.com/
General problem
The choice of suitable reference genes is absolutely crucial in RT-
qPCR gene expression analysis. Often, genes from commercial panels
don't work well for one's own biological context. Ideally, the
expression of reference genes should remain unchanged across samples
within the context under study.
Solution
RefGenes is an online app from Genevestigator that allows users to
search for genes that are most stable across a chosen set of samples
based on microarray data. This set of samples can be chosen according
to experimental conditions or tissue types. For example, if you are
performing a RT-qPCR experiment on mouse liver samples, you can use
RefGenes to identify the set of genes that are most stable across all
microarrays done on mouse liver in Genevestigator. This method offers
two major improvements over existing methods because a) it does not
narrow down from a small set of genes (e.g. commercial housekeeping
gene panels), but looks for novel candidates from a genome-wide set of
genes b) it is based on condition-specific stability. The below schema
shows how RefGenes can be used in combination with existing approaches
to yield valuable reference genes for specific experimental
conditions.
RefGenes: identification of reliable and condition specific reference
genes for RT-qPCR data normalization.
Hruz T, Wyss M, Docquier M, Pfaffl MW, Masanetz S, Borghi L, Verbrugge
P, Kalaydjieva L, Bleuler S, Laule O, Descombes P, Gruissem W and P
Zimmermann
BMC Genomics 2011, 12: 156
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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..... http://www.***.com/
Over 2,000 downloads up to now!
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qPCR 2011 US - http://www.***.com/
Mission Center Ballroom, Santa Clara Convention Center, 5001 Great
America Parkway, Santa Clara, CA 95054
Symposium Focus
* Preanalytics, Standardization and quality control
* High throughput expression profiling digital PCR
* Epigenetics, mutation analysis and copy number variation
* Molecular diagnostics of complex diseases detection and profiling
of tumor cells
* Single-cell and subcellular expression profiling
* Non coding RNAs
* qPCR in Forensics and AgriBio
* Next Generation Sequencing (NGS)
* qPCR & NGS experimental design and data mining
* Clinical applications of qPCR and NGS
Follow the symposium agenda! http://www.***.com/
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Please forward this qPCR NEWS
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to further scientists and friends who are interested in qPCR !
Best regards,
Michael W. Pfaffl
responsible Editor of the Gene Quantification Pages
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qPCR NEWS - October 2011 - focus on MIQE and RefGenes