qPCR NEWSLETTER - October 2007

Editor www.Gene-Quantification.info via methods%40net.bio.net (by editor from gene-quantification.info)
Fri Oct 26 04:32:04 EST 2007

qPCR NEWSLETTER - October 2007

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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:

- Update of real-time RT-PCR normalisation page

- New papers about RNA integrity testing

- NARG 2007 qPCR survey results

- Download the talk and poster PDFs from the qPCR 2007 Event

- qPCR Symposium USA next week San Francisco

- qPCR application workshops in 2007 and 2008


Data normalisation in real-time RT-PCR is a further major step in gene
quantification analysis (Bustin 2002, Pfaffl 2001 ). The reliability
of any relative RT-PCR experiment can be improved by including an
invariant endogenous control (reference gene) in the assay to correct
for sample to sample variations in RT-PCR efficiency and errors in
sample quantification. A biologically meaningful reporting of target
mRNA copy numbers requires accurate and relevant normalisation to some
standard and is strongly recommended in kinetic RT-PCR. But the
quality of normalized quantitative expression data cannot be better
than the quality of the normalizer itself. Any variation in the
normalizer will obscure real changes and produce artifactual changes
(Bustin 2000).  Real-time RT-PCR-specific errors in the quantification
of mRNA transcripts are easily compounded with any variation in the
amount of starting material between the samples, e.g. caused by sample-
to-sample variation, variation in RNA integrity, RT efficiency
differences and cDNA sample loading variation (Stahlberg 2003 2004a,
2004b). This is especially relevant when the samples have been
obtained from different individuals, different tissues and different
time courses, and will result in the misinterpretation of the derived
expression profile of the target genes. Therefore, normalisation of
target gene expression levels must be performed to compensate intra-
and inter-kinetic RT-PCR variations (sample-to-sample and run-to-run
variations) (Pfaffl & Hageleit 2001).

Find more info on our web page:   http://normalisation.gene-quantification.info/


Universal reference method for real-time.
Neil Ivan Bower, Ralf Joachim Moser, Jonathan Robert Hill, and Sigrid
Arabella Lehnert
BioTechniques (2007) 42: 199-206

Validation of rat reference genes for improved quantitative gene
expression analysis using low density arrays.
Jenny Hong Cai, Shibing Deng, Steven W. Kumpf, Patricia A. Lee,
Panayiotis Zagouras, Anne Ryan, and Dan S. Gallagher
BioTechniques (2007) 42: 503-512

In search of suitable reference genes for gene expression studies of
human renal cell carcinoma by real-time PCR.
Monika Jung, Azizbek Ramankulov, Jan Roigas, Manfred Johannsen, Martin
Ringsdorf, Glen Kristiansen & Klaus Jung
BMC Molecular Biology (2007) 8: 47

GeneChip, geNorm, and gastrointestinal tumors: novel reference genes
for real-time PCR.
Mark Kidd, Boaz Nadler, Shrikant Mane, Geeta Eick, Maximillian
Malfertheiner, Manish Champaneria, Roswitha Pfragner, and Irvin Modlin
Physiol Genomics (2007) 30:  363–370

Developmental expression profiles of Xenopus laevis reference genes.
Sindelka R, Ferjentsik Z, Jonak J.
Dev Dyn. (20069  235(3): 754-758

Statistical Selection of Maintenance Genes for Normalization of Gene
Yifan Huang Jason C. Hsu† Mario Peruggia‡ Abigail A. Scott
Statistical Applications in Genetics and Molecular Biology (2006) 5(1)

Quantification of cDNA generated by reverse transcription of total RNA
provides a simple alternative tool for quantitative RT-PCR
Jiri Libus and Helena Štorchová
Institute of Experimental Botany, Prague, Czech Republic
BioTechniques (2006) 41: 156-164

Equivalence test in quantitative reverse transcription polymerase
chain reaction: confirmation of referencegenes suitable for
normalizationHaller F, Kulle B, Schwager S, Gunawan B, von Heydebreck
A, Sultmann H, Fuzesi L.Anal Biochem. (2004) 335(1): 1-9

Find more publication on our web page:   http://normalisation.gene-quantification.info/


REVIEW:  RNA integrity and the effect on the real-time qRT-PCR

Molecular Aspects of Medicine 27 (2006) 126–139
Simone Fleige & Michael W. Pfaffl
Physiology Weihenstephan, Center of Life and Food Sciences (ZIEL),
Technical University of Munich, 85350 Freising, Germany;  TATAA
Biocenter Germany, Freising-Weihenstephan, Germany


The assessment of RNA integrity is a critical first step in obtaining
meaningful gene expression data. Working with low-quality RNA may
strongly compromise the experimental results of downstream
applications which are often labour-intensive, time-consuming, and
highly expensive. Using intact RNA is a key element for the successful
application of modern molecular biological methods, like qRT-PCR or
micro-array analysis. To verify RNA quality nowadays commercially
available automated capillary-electrophoresis systems are available
which are on the way to become the standard in RNA quality assessment.
Profiles generated yield information on RNA concentration, allow a
visual inspection of RNA integrity, and generate approximated ratios
between the mass of ribosomal sub-units. In this review, the
importance of RNA quality for the qRT-PCR was analyzed by determining
the RNA quality of different bovine tissues and cell culture.
Independent analysis systems are described and compared (OD
measurement, NanoDrop, Bioanalyzer 2100 and Experion). Advantage and
disadvantages of RNA quantity and quality assessment are shown in
performed applications of various tissues and cell cultures. Further
the comparison and correlation between the total RNA integrity on PCR
performance as well as on PCR efficiency is described. On the basis of
the derived results we can argue that qRT-PCR performance is affected
by the RNA integrity and PCR efficiency in general is not affected by the
RNA integrity. We can recommend a RIN higher than five as good total
RNA quality and higher than eight as perfect total RNA for downstream



Newly added RNA intergrity publication:

Preanalytical mRNA Stabilization of Whole Bone Marrow Samples
Technical Advance:  Isolation of Microarray-Grade Total RNA, MicroRNA,
and DNA from a Single PAXgene Blood RNA Tube
RNA quality in frozen breast cancer samples and the influence on gene
expression analysis - a comparison of three evaluation methods using
microcapillary electrophoresis traces.
Tissue preparation for laser capture microdissection and RNA
extraction from fresh frozen breast tissue.
Simultaneous control of DNA and RNA processing efficiency using a
nucleic acid calibration set.
Einfluss der RNA Integrit auf die quantitative real-time RT-PCR  (in


qPCR 2007

Link to the qPCR 2007 Event homepage:   http://qpcr2007.gene-quantification.info/
3rd International qPCR Symposium & Application Workshop in Freising -
Weihenstephan 2007

Download  =>  qPCR 2007 Symposium Proceedings   ISBN-13:

Download  =>  qPCR 2007 Talks and Posters  (now available)


Download the talks and poster presentations from previous qPCR

- qPCR 2004 Event downloads

- qPCR 2005 Event downloads

- Leipzig 2005 Meeting downloads


Upcoming Events World-wide academic and commercial qPCR Events

Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars,
qPCR Education Program, ...etc...
Please submit your qPCR event here  =>  events from gene-


qPCR Symposium USA

Symposium Focus:
Markers, Stem Cells, Single Cell, siRNA, miRNA, Diagnostics, Immuno-
qPCR, Expression Profiling,
Poster Presentation, Workshops in qPCR



TATAA Biocenter Germany - qPCR Application workshops

At the TATAA Biocenter Germany we offer qPCR application workshops,
the 3-day Core Module and a 2-day Biostatistics Module.  qPCR courses
are held in regularly in Göteborg, 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 courses are held in cooperation with the Institute
of Physiology, located at the Technical University of Munich, in
Freising-Weihenstephan, near Munich, very close to the Munich Airport
(MUC). For more information and to register for the qPCR application
workshops, please see our web page:  http://tataa.gene-quantification.info/

Course Occasions 2007 and 2008:
3-day qPCR Core Module  (Mon. - Wed.)    and     2-day BioStatistics
Module (Thu. - Fri.)

*   26 - 30th November 2007 (in Freising, Germany, Kurs wird in
DEUTSCH gehalten, German language)
*   3rd - 7th March 2008  (in Freising, Germany, English language)
*   5 - 9th May 2008 (in Freising, Germany, Kurs wird in DEUTSCH
gehalten, German language)
*   7 - 11th July 2008  (in Freising, Germany, English language)

Please register here  =>  http://www.tataa.com/Courses/Courses.html


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


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