qPCR NEWS April 2009 - focus on microRNA normalisation

qPCR NEWS April 2009 - focus on microRNA normalisation
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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 in new microRNA reviews
- NEW section about normalisation in microRNA experiments =>
[Only registered users see links. ]
- Online translation service of the Gene Quantification
- New qPCR events in autumn 2009
- New qPCR workshop modules at the TATAA Biocenter Germany
- Online translation service of the Gene Quantification =>
[Only registered users see links. ]

New qPCR workshop modules at the TATAA Biocenter Germany =>
[Only registered users see links. ]
European wide qPCR application workshops => register now !
=> course program spring - summer 2009
=> [Only registered users see links. ]


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Data normalisation in microRNA experiments using qRT-PCR is a new
challenge in gene quantification analysis. 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 the qRT-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 microRNA qRT-PCR.

=> But the quality of normalized quantitative expression data cannot
be better than the quality of the normalizer itself.
=> Which are the best endogen microRNA normalizers?
=> Can we apply the same "microRNA normalizing strategy"?

Any variation in the normalizer will obscure real changes and produce
artifactual changes. Real-time RT-PCR-specific errors in the
quantification of microRNA transcripts are easily compounded with any
variation in the amount of starting material between the samples, e.g.
caused by sample-to-sample variation and cDNA sample loading
variation. 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 & run-to-run variations).

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 microRNA or 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. It
requires an accurate quantification of the isolated total RNA or mRNA
or microRNA fraction by optical density at 260 nm, Lab-on-Chip
capillary electrophoresis instruments, or Ribogreen RNA Quantification
Kit.

To normalize the absolute amount according to a single reference gene
(or better a set of multiple stable reference genes), further sets of
kinetic PCR reactions has to be performed for the invariant endogenous
control(s) 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
biological samples can then directly be compared.

The workflow:

- check for good RNA integrity (=> [Only registered users see links. ])
- select stable internal reference microRNA or suitable smallRNAs (via
Genorm or Normfinder)
- calculate reference-gene-index of selected normalizers (geometric
mean of Cq)
- apply relative quantification strategy (comparable to mRNA relative
quantification)
- apply PCR efficiency correction (if wanted)
- for microRNA normalistion strategies see papers below
- or find some more ideas here => [Only registered users see links. ]
=> [Only registered users see links. ]


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- Identification by Real-time PCR of 13 mature microRNAs
differentially expressed in colorectal cancer and non-tumoral
tissues.
- Normalization of microRNA expression levels in quantitative RT-PCR
assays: identification of suitable reference RNA targets in normal and
cancerous human solid tissues.
- Identification of suitable endogenous control genes for microRNA
gene expression analysis in human breast cancer.
- High-throughput stem-loop RT-qPCR miRNA expression profiling using
minute amounts of input RNA.
- Facile means for quantifying microRNA expression by real-time PCR.
- A single-molecule method for the quantitation of microRNA gene
expression.
- Endogenous Controls for Real-Time Quantitation of miRNA Using
TaqMan® MicroRNA Assays.


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online translation

Since October 2008 we provide an online translation service of the
Gene Quantification pages to several languages. Please recognize this
is an automatic and robotic based translation service, and therefore
we can give NO guarantee about the generated content. It should help
to understand the "rough" content of the Gene Quantification pages,
but still the original is the ENGLISH version:

[Only registered users see links. ]


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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 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 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:
=> [Only registered users see links. ]


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.)


20 - 22 April 2009 (E) NEW microRNA qPCR

11 - 15 May 2009 (Deutsch)
3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics Module
(Thu. - Fri.)

15 - 19 Juni 2009 (E)
3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics Module
(Thu. - Fri.)

13 - 15 Juli 2009 (E) NEW microRNA qPCR

27 - 31 Juli 2009 (E)
3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics Module
(Thu. - Fri.)

=> [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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