qPCR NEWSLETTER - April 2010 - microRNA normalisation & QC

Editor www.Gene-Quantification.info via methods%40net.bio.net (by editor from gene-quantification.info)
Thu Apr 29 05:10:19 EST 2010

qPCR NEWSLETTER - April 2010 - microRNA normalisation & QC

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:

- microRNA quality control
- microRNA normalisation
- qPCR Application Workshops  -  and more ... ... ...

European wide qPCR application workshops =>  register now !
Download course brochure 2010 => http://www.gene-quantification.de/bioeps-course-programm-2010.pdf
Register here => http://site.bioeps.com/index.php?option=com_seminar&Itemid=6

Updated section  -  microRNA normalisation in real-time qRT-PCR


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 a comparable normalising strategy as done for mRNAs ?

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

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 total RNA integrity
- 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
- apply PCR efficiency correction (if wanted)
- for microRNA normalistion strategies see papers below
- or find some more ideas in the Relative Quantification Section

some selected publications microRNA normalisation in qRT-PCR:

- Overview and workflow in microRNA normalisation
- Identification by Real-time PCR of 13 mature microRNAs
differentially expressed in colorectal cancer and non-tumoral tissues
- Expression profiling of microRNA using real-time quantitative PCR,
how to use it and what is available
- A novel and universal method for microRNA RT-qPCR data normalization
- Systematic comparison of microarray profiling, real-time PCR, and
next-generation sequencing technologies for measuring differential
microRNA expression
- A modified LOESS normalization applied to microRNA arrays:  a
comparative evaluation
- Improved microRNA quantification in total RNA from clinical samples
- Normalization of microRNA expression levels in quantitative RT-PCR
assays:  Identification of suitable reference RNA targets in normal
and cancerous human solid tissues
- Measuring microRNAs: comparisons of microarray and quantitative PCR
measurements, and of different total RNA prep methods
- 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
- Endogenous Controls for Real-Time Quantitation of miRNA Using
TaqMan® MicroRNA Assays
- microRNA normalisation in array experiments:

Quality assessment and data analysis for microRNA expression arrays:

- A comparison of normalization techniques for microRNA microarray
- A sensitive array for microRNA expression profiling (miChip) based
on locked nucleic acids (LNA)
- miChip: an array-based method for microRNA expression profiling
using locked nucleic acid capture probes
- A personalized microRNA microarray normalization method using a
logistic regression model
- Intra-platform repeatability and inter-platform comparability of
microRNA microarray technology
- Impact of normalization on miRNA microarray expression profiling
- Comparison of normalization methods with microRNA microarray

=>  http://miRnorm.gene-quantification.info


BioEPS GmbH - qPCR Application Workshops

Life Science is still a growing sector and new methods and
technologies are continously developed. Therefore permanent training
and education becomes so important.
With our specific course program we are offering a range of high-
quality course modules, in cooperation with different companies to
give a general and independent overview of existing qPCR technologies
and systems. Our course issues are based on skilled know-how from own
research studies and publications.
Our aim is to point out a critical way of thinking to increase the
quality and outcome of experimental data.
All BioEPS services are based on the MIQE guidelines (Minimum
Information for Publication of Quantitative real-time PCR
Experiments), the first guidelines for quality assurance within qPCR
technology  =>  MIQE.gene-quantification.info


All courses are held regularly in Freising-Weihenstephan, Germany, in
German and English language.
Further customized workshops and specialized trainings will be held as
well across Europe and world-wide.
Workshops are powered by 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 =>

Course Occasions 2010:

3-day qPCR Basic Module
2-day BioStatistics & Expression Profiling Module
3-day single-cell qPCR
2-day microRNA qPCR
1-day HRM
2-das qPCR-R data analysis   NEW !
1-day Project Management   NEW !
2-day Quality Management  NEW !

Download course brochure 2010 => http://www.gene-quantification.de/bioeps-course-programm-2010.pdf
Register here => http://site.bioeps.com/index.php?option=com_seminar&Itemid=6
Access to our workshops => http://www.gene-quantification.de/bioeps-access.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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