qPCR NEWSLETTER - July 2007

qPCR NEWSLETTER - July 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:

- A lot new publications around the normalisation procedure appeared
during the last years:
- Normalisation - Reference Genes - Housekeeping Genes
- Update of our Gene Quantification Page Directory
- Update of the qPCR Webinar page
- qPCR application workshops in autumn in Germany
- qPCR Symposium USA in October/November 2007

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Normalisation - Reference Genes - Housekeeping Genes

page 1 - [Only registered users see links. ]
page 2 - [Only registered users see links. ]
page 3 - [Only registered users see links. ]

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).

Data normalisation can be carried out against an endogenous
unregulated reference gene transcript or against total cellular DNA or
RNA content (molecules/g total DNA/RNA and concentrations/g total DNA/
RNA). Normalisation according the total cellular RNA content is
increasingly used, but little is known about the total RNA content of
cells or even about the mRNA concentrations. The content per cell or
per gram tissue may vary in different tissues in vivo, in cell culture
(in vitro), between individuals and under different experimental
conditions. Nevertheless, it has been shown that normalisation to
total cellular RNA is the least unreliable method (Bustin 2000 Bustin
2002). It requires an accurate quantification of the isolated total
RNA or mRNA fraction by optical density at 260 nm, Agilent Bioanalyser
2100, or Ribogreen RNA Quantification Kit. Alternatively the rRNA
content has been proposed as an optimal and stable basis for
normalisation, despite reservations concerning its expression levels,
transcription by a different RNA polymerase and possible imbalances in
rRNA and mRNA fractions between different samples (=> RNA and RT ).

To normalize the absolute quantification according to a single
reference gene, a second set of kinetic PCR reactions has to be
performed for the invariant endogenous control on all experimental
samples and the relative abundance values are calculated for internal
control as well as for the target gene. For each target gene sample,
the relative abundance value obtained is divided by the value derived
from the control sequence in the corresponding target gene. The
normalized values for different samples can then directly be compared
(Pfaffl 2001 and => relative expression).

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A lot new publications around the normalisation procedure appeared
during the last years => please have a look!
Normalization in real-time PCR - page 3 - [Only registered users see links. ]

Development of a new set of reference genes for normalization of real-
time RT-PCR data of porcine backfat and longissimus dorsi muscle, and
evaluation with PPARGC1A.

Selection of reference genes for quantitative real-time PCR in bovine
preimplantation embryos.

Generic normalization method for real-time PCR Application for the
analysis of the mannanase gene expressed in germinating tomato seed.

Simultaneous control of DNA and RNA processing efficiency using a
nucleic acid calibration set.

Universal reference method for real-time PCR gene expression analysis
of preimplantation embryos.

Development and evaluation of canine reference genes for accurate
quantification of gene expression.

Genome-Wide Identification and Testing of Superior Reference Genes for
Transcript Normalization in Arabidopsis.

Normalizing genes for quantitative RT-PCR in differentiating human
intestinal epithelial cells and adenocarcinomas of the colon.

Statistical Selection of Maintenance Genes for Normalization of Gene
Expressions.

Evaluation of Reference Genes for Studies of Gene Expression in Human
Adipose Tissue.

Validation of rat reference genes for improved quantitative gene
expression analysis using low density arrays.

A Relevant Reference Gene and Normalization for mRNA Real-Time PCR
Quantification in Specimens with Distinct Cell Types and Variant
Integrity.

Quantification of cDNA generated by reverse transcription of total RNA
provides a simple alternative tool for quantitative RT-PCR
normalization.

Error propagation in relative real-time reverse transcription
polymerase chain reaction quantification models: The balance between
accuracy and precision.

Selection of reference genes in mouse embryos and in differentiating
human and mouse ES cells.

Sex-dependent expression of seven housekeeping genes in rat liver.

QuantitativeMulti-Gene Expression Profiling of Primary Prostate
Cancer.

Selection of reference genes for quantitative RT-PCR studies in
striped dolphin (Stenella coeruleoalba) skin biopsies.

Genomic DNA standards for gene expression profling in Mycobacterium
tuberculosis.

Housekeeping gene selection for real-time RT-PCR normalization in
potato during biotic and abiotic stress.

Normalization in real-time PCR - page 3 - [Only registered users see links. ]


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TALKS Update of the qPCR Talk and Webinar page

A lot of interesting TALKs, WEBINARs, SLIDE SHOWs, and PODCASTs from
various speakers, biotec companies, qPCR Events, and international
journals (Nature and Science) are FREE for download. Have a look and
you will definitely something interesting for your scientific work !
[Only registered users see links. ]
[Only registered users see links. ]

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INFOPORTAL Protocols on qPCR INFOPORTAL

[Only registered users see links. ]

Protocols online is a resource for protocols, including authoritative,
peer-reviewed 'protocols and an interactive network.

NEW => qPCR and Molecular Biology - Discussion Forums

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Upcoming Events World-wide academic and commercial qPCR Events
[Only registered users see links. ]

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


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qPCR Symposium USA
[Only registered users see links. ]

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

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WORKSHOP


Opening Opening of TATAA Biocenter, Prague => [Only registered users see links. ]
Real time open qPCR course from 21th - 25th May 2007 => download
flyer


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:

[Only registered users see links. ]

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

24 - 28th September 2007 (in Freising, Germany, Kurs wird in DEUTSCH
gehalten, German language)
22 - 26th October 2007 (in Freising, Germany, English language)
26 - 30th November 2007 (in Freising, Germany, English language)
Please register here => [Only registered users see links. ]



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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
[Only registered users see links. ]

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