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:
- RNA integrity
- various RNA quality related papers
- DEGRADOMETER software
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REVIEW: RNA integrity and the effect on the real-time qRT-PCR
[Only registered users see links. ]
The assessment of RNA integrity is a critical ?rst 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. Pro?les
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 effciency is described. On the basis of
the derived results we can argue that qRT-PCR performance is a?ected by
the RNA integrity and PCR effciency in general is not affected by the
We can recommend a RIN higher than ?ve as good total RNA quality and
higher than eight as perfect total RNA for downstream application.
- Towards standardization of RNA quality assessment using
user-independent classifiers of microcapillary electrophoresis traces.
- Effect of Fixatives and Tissue Processing on the Content and
Integrity of Nucleic Acids.
- Pitfalls of quantitative real-time reverse-transcription polymerase
chain reaction (with special focus on RNA quality).
- Real-time, fluorescence-based quantitative PCR: a snapshot of current
procedures and preferences.
- Comparison of relative mRNA quantification models and the impact of
RNA integrity in quantitative real-time RT-PCR.
- Assessment of fixatives, fixation, and tissue processing on
morphology and RNA integrity.
- TaqMan PCR assay in the control of RNA normalization in human
post-mortem brain tissue.
- RNA integrity as a quality indicator during the first steps of RNP
purifications : A comparison of yeast lysis methods.
- Modification of the Standard Trizol-Based Technique Improves the
Integrity of RNA Isolated from RNase-Rich Placental Tissue.
- Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical
- The External RNA Control Consortium: a progress report.
- The RIN: an RNA integrity number for assigning integrity values to
- Performance Comparison of the Experion Automated Electrophoresis
System and a Competing Automated System for RNA Analysis (Bio-Rad).
- The RNA Integrity Number (RIN) (Agilnet Technologies)
- RNA Integrity Number (RIN) - Standardization of RNA Quality Control
- The importance of RNA quality in Real Time Quantitaive RT-PCR - Eric
Lander, Ambion, ABRF 2002
- RNA Integrity Number (RIN) - Standardization of RNA Integrity
Measurements (talk at the 1st qPCR Event by Odilo Müller)
- RNA Quality and Yields from Frozen Tissues.
- Working with Fixatives, Fixation and Tissue Processing to improve
Morphology and RNA Quality - Melissa Cox, Pfizer
- External RNA Quality Control by RT-PCR - by Koch et al.
- Degradometer Manual
- Download Degradometer software version 1.41.zip
- PAPER: Chipping away at the chip bias: RNA degradation in microarray
- POSTER: Estimation and Reduction of the Bias Caused by RNA
Degradation in Microarray Analysis.
Measurements of gene expression are based on the assumption that the
analyzed RNA sample closely resembles the amount of transcripts in
vivo. Established knowledge that transcripts of different genes possess
different stabilities suggests that degradation of RNA occurring during
the isolation procedure is also non-uniformly distributed among
different RNA species. Indeed, comparison of RNA samples of different
degrees of degradation shows that up to 75% of microarray-based
measurements of differential gene expression can be caused by
degradation bias alone1. We demonstrate that analysis of
capillary-electrophoresis data does allow reproducible characterization
of RNA degradation and its differentiation from apoptosis-associated
RNA cleavage. Degradometer software for quantification of RNA integrity
is available on our website ([Only registered users see links. ]). Our results suggest
that comparison of RNA samples of similar integrity eliminates skewed
results of differential gene expression. Consequently, information
about quantification of RNA integrity will help to improve
reproducibility of microarray results.
TATAA Biocenter have found that the worldwide demand for training in
the field of quantitative real-time PCR (qPCR) is huge. To coordinate
this we aim to arrange practical courses, e.g. 3-day Core Module and
1-day or 2-day Biostatistics Module:
The basic qPCR Core Module contains three workshop days:
First workshop day will be directed to people planning or considering
using qPCR in their research and also users not yet fully familiar will
quantitative PCR. Second day targets more advanced users and people
concentrating on different quantification strategies. The third day
focuses on aspects in sample preparation and reverse transcription. The
additional qPCR Biostatistics Module explains statistics applicable to
qPCR and teaches how to use statistics to interpret qPCR gene
expression data, and classify samples based on qPCR expression
Courses contain both, theoretical seminars and practical hands-on
training with experienced supervision. Practical training will be
performed on three different real-time PCR cyclers, using multiple
detection chemistries. The Biostatistics Module is further based on
computer-based demonstrations. Please bring your own Laptop !
qPCR courses are held in regularly in Göteborg, Sweden and in
Freising-Weihenstephan, Germany (near Munich, very close to the Munich
Airport - MUC). Depending on the occasion different prices may apply.
Also different course modules are available on the different occasions.
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.
Course Occasions 2006:
23rd - 27th October 2006 ( BioStat Module is still booked )
20th - 24th November 2006 ( both Modules are still available )