qPCR NEWS May 2009 - focus on RNA integrity
qPCR NEWS May 2009 - focus on RNA integrity
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Dear researcher,
dear Gene Quantification page reader,
Our newsletter informs about the latest news in quantitative real-time
PCR (qPCR and qRT-PCR), which are compiled and summarised on the Gene
Quantification homepage. The focus of this newsletter issue is:
- The MIQE Guidelines Minimum Information for Publication of
Quantitative Real-Time PCR Experiments
- RNA integrity - latest papers
- PowerNest - illuminating error in qPCR experiment design
- new TAG CLOUD for better navigation =3D> http://directory.gene-quantifica=
tion.info/
- New qPCR workshop modules at the TATAA Biocenter Germany =3D>
http://tataa.gene-quantification.info/
- European wide qPCR application workshops =3D> register now !
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The MIQE Guidelines Minimum Information for Publication of
Quantitative Real-Time PCR Experiments
Stephen A. Bustin, Vladimir Benes, Jeremy A. Garson, Jan Hellemans,
Jim Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W.
Pfaffl, Gregory L. Shipley, Jo Vandesompele, & Carl T. Wittwer
Clinical Chemistry 2009, 55(4): 611-622
=3D> http://www.gene-quantification.de/miqe-bustin-et-al-clin-chem-2009.pdf
BACKGROUND: Currently, a lack of consensus exists on how best to
perform and interpret quantitative real-time PCR (qPCR) experiments.
The problem is exacerbated by a lack of sufficient experimental detail
in many publications, which impedes a reader's ability to evaluate
critically the quality of the results presented or to repeat the
experiments.
CONTENT: The Minimum Information for Publication of Quantitative Real-
Time PCR Experiments (MIQE) guidelines target the reliability of
results to help ensure the integrity of the scientific literature,
promote consistency between laboratories, and increase experimental
transparency. MIQE is a set of guidelines that describe the minimum
information necessary for evaluating qPCR experiments. Included is a
checklist to accompany the initial submission of a manuscript to the
publisher. By providing all relevant experimental conditions and assay
characteristics, reviewers can assess the validity of the protocols
used. Full disclosure of all reagents, sequences, and analysis methods
is necessary to enable other investigators to reproduce results. MIQE
details should be published either in abbreviated form or as an online
supplement.
SUMMARY: Following these guidelines will encourage better
experimental practice, allowing more reliable and unequivocal
interpretation of qPCR results.
=3D> http://miqe.gene-quantification.info/
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As part of the MIQE guidelines - chapter 5.1 - the RNA quality control
is an essential step in the quantification process.
5. Nucleic acid quality control
5.1. RNA samples
Since it is advisable to use approximately the same amount of RNA for
cDNA synthesis when comparing different samples, quantification of RNA
in extracted samples is important. However, there are several
procedures in common use, including spectrophotometry (Nanodrop),
microfluidic analysis (Agilent BioAnalyser, BioRad Experion),
capillary gel electrophoresis (Qiagen QIAexcel) or fluorescent dye
detection (Ribogreen); all produce different results making it unwise
to compare data obtained using the different methods. The preferred
method for RNA quantity determination uses fluorescent RNA binding
dyes, for example RiboGreen, which are best for the detection of low
target concentrations. In any case, it is advisable to measure all
samples using one method only and to report this
information. ... ... ...
latest papers =3D> http://www.gene-quantification.de/rna-integrity2.html
- Reverse transcription-quantitative polymerase chain reaction:
description of a RIN-based algorithm for accurate data normalization.
- Time course analysis of RNA stability in human placenta.
- Evaluation of isolation methods and RNA integrity for bacterial RNA
quantitation.
- A comparison and evaluation of RNA quantification methods using
viral, prokaryotic, and eukaryotic RNA over a 10(4) concentration
range.
- Validation of lab-on-chip capillary electrophoresis systems for
total RNA quality and quantity control.
- Improved RNA quality and TaqMan Pre-amplification method (PreAmp) to
enhance expression analysis from formalin fixed paraffin embedded
(FFPE) materials.
- Optimization of the method of RNA isolation from paraffin blocks to
assess gene expression in breast cancer.
- Measuring microRNAs: comparisons of microarray and quantitative PCR
measurements, and of different total RNA prep methods.
- Focus on RNA isolation: obtaining RNA for microRNA (miRNA)
expression profiling analyses of neural tissue.
- Systematic analysis of microRNA expression of RNA extracted from
fresh frozen and formalin-fixed paraffin-embedded samples.
- Stability of RNA isolated from post-mortem tissues of Atlantic
salmon (Salmo salar L.)
- Prediction of qualitative outcome of oligonucleotide microarray
hybridization by measurement of RNA integrity using the 2100
Bioanalyzer capillary electrophoresis system.
- Removal of contaminating DNA from commercial nucleic acid extraction
kit reagents.
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PowerNest - illuminating error in qPCR experiment design
PowerNest is a software tool enabling experimenters to explore the
effect of sampling on noise propagation throughout qPCR assays. The
sampling process is assumed to be comprised of a number of levels; the
acquisition of a sample and the preparation of extracted material,
reverse-transcription of the mRNA, and the qPCR itself. Given a small
set of data, representative of a larger assay, the error at each stage
of the experiment is profiled using a nested-ANOVA.
Armed with this information, PowerNest allows the experimenter to
explore the effects of modifications to the experimental design on the
expected total error of the assay. When given the financial cost of
replicates at each level, PowerNest will calculate a cost-optimal
sampling-plan, delivering an experiment design that will minimise
processing error and maximise the statistical resolution of the assay.
The software is temporarily undergoing final testing, during which
time it has been made available as a free download =3D>
http://www.gene-quantification.de/main-bioinf.shtml#powernest
PowerNest Poster =3D> http://www.gene-quantification.de/powernest-poster.pd=
f
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Upcoming Events World-wide academic and commercial qPCR Events
http://events.gene-quantification.info/
Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars,
qPCR Education Program, etc.
Please submit your qPCR event here =3D> events@gene-
quantification.info
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qPCR WORKSHOP
BioEPS GmbH / TATAA Biocenter Germany - qPCR Application workshops
At the TATAA Biocenter Germany we offer qPCR application workshops, a
3-day qPCR Core Module and a 2-day qPCR Biostatistics Module. All
courses are held regularly in G=F6teborg, Sweden, in English and in
Freising-Weihenstephan, Germany, in German and English, and in Prague,
Czech Republic in English and Czech.
Depending on the occasion the workshop language and the different
prices may apply. Further customized workshops and specialized
trainings will be held as well across Europe and world-wide.
TATAA Biocenter Germany workshops are held in cooperation with BioEPS
GmbH, located at the campus of the Technical University of Munich, in
Freising-Weihenstephan, very close to the Munich Airport (MUC). For
more information and registration, please see our web page:
=3D> http://TATAA.gene-quantification.info/
Course Occasions 2009:
3-day qPCR Core Module (Mon. - Wed.)
2-day BioStatistics Module (Thu. - Fri.)
3-day single-cell qPCR Module (Mon. - Wed.)
3-day microRNA Module (Mon. - Wed.)
15 - 19 Juni 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day
BioStatistics (Thu. - Fri.)
13 - 15 Juli 2009 (E) NEW microRNA qPCR (in cooperation with
Qiagen)
27 - 31 Juli 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day
BioStatistics (Thu. - Fri.)
14 - 16 September 2009 (E) NEW single-cell qPCR
19 - 23 September 2009 (E) 3-day microRNA Module (Mon. - Wed.) & 2-
day BioStatistics (Thu. - Fri.)
26 - 28 October 2009 (E) 3-day qPCR Core Module (Mon. - Wed.)
16 - 20 November 2009 (E) 3-day microRNA Module (Mon. - Wed.) & 2-
day BioStatistics (Thu. - Fri.)
7 - 11 December 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-
day BioStatistics (Thu. - Fri.)
=3D> http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pd=
f
=3D> http://www.gene-quantification.de/bioeps-courses-autumn-2009.pdf
=3D> http://www.gene-quantification.de/single-cell-qpcr-course-sept-2009.pd=
f
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Forward Please send the qPCR NEWS to further scientists and friends
who are interested in qPCR !
Best regards,
Michael W. Pfaffl
responsible Editor of the Gene Quantification Pages
http://www.gene-quantification.info
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