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qPCR 2007 Symposium POSTER
Presentations
Main Session:
microRNA
– siRNA Applications
Poster
number P 001 – P
009 Location:
Student Cafeteria P 001 Regulation
of cadmium-induced responses in Arabidopsis
thaliana: The role of microRNAs. Smeets K., Cuypers A.,
Donckers K., Remans T., Vangronsveld J. Centre for
environmental sciences, Email:
karen.smeets@uhasselt.be As a highly
toxic metal, knowledge
about cadmium-induced pathways in plants is rather scarce. Cadmium does
appear
to provoke several oxidative stress related effects, similar to other
heavy
metals. Because of its non-redox active nature, the induced redox
disequilibrium has to be established via indirect pathways. A specific
role of
this increased ROS-content in the regulation in cadmium-induced
responses,
however, remains to be elucidated. Therefore,
the early effects of
cadmium toxicity were examined by performing a complete transcriptome
analysis
in Arabidopsis thaliana in combination with the analysis of
specific
cadmium-induced microRNAs. From these results, a highly coordinated
expression
response can be concluded during moderate cadmium toxicity, in which an
increased ROS-content could play a central role. Furthermore, the
induced/inhibited regulative genes are already responding after a few
hours of
exposure and expression is well regulated between the different organs. In this study
environmental
realistic exposure concentrations were used, therefore the outcome is
highly
relevant for further research on heavy metal contamination. P
002 Efficient
and specific quantification of mammalian
microRNAs using a novel real-time PCR approach. Martin Kreutz1,
James Qin1,
Holger Engel 2, Po-Jen Shih1, Martin
Schlumpberger 2, Subrahmanyam Yerramilli1,
and Eric Lader1 1QIAGEN
Sciences, Email:
martin.schlumpberger@qiagen.com We have
developed an efficient and
accurate method for transcriptome-wide miRNA quantification using a
SYBR Green
based, real-time PCR detection system. The miScript System is highly
specific
and sensitive, and requires very small amounts of input RNA. The system
enables
detection of miRNAs as well as mRNAs using the same cDNA preparation
allowing
simultaneous quantification of miRNA and target mRNA. A single cDNA
prep is sufficient for
quantification of several miRNAs of interest, avoiding the need to
prepare
multiple cDNA preps to quantify each miRNA. Use of a single prep
eliminates
potential variation that could arise during multiple cDNA synthesis
reactions.
We will demonstrate the application of this technology to miRNA
expression
profiling in a model human cell-culture system. This
technology offers researchers a
sensitive, specific, and easy-to-perform approach for accurate
expression
profiling of miRNAs using a small amount of total RNA containing
miRNAs. A
single cDNA prep from a precious RNA sample is sufficient for profiling
of all
the known miRNAs in a given model system. This is a substantial advance
in the
state of the art and would be of broad interest to all scientists
studying
miRNAs and their targets P
003 Measure
of miRNAs in microdissected colorectal tumor
tissues: optimization of RNA preparation. Stefania Gelmini, Marta Tarter,
Francesca Salvianti, Claudio Orlando, Lisa Simi, Mario Pazzagli, Pamela
Pinzani. Clinical
Biochemistry Unit, Dept. of
Clinical Physiopathology, Email:
s.gelmini@dfc.unifi.it MicroRNAs
(miRNAs) are short
non-coding RNA molecules involved in gene expression regulation by
repressing
translation or cleaving RNA transcripts. Recently a connection between
miRNAs
and cancer was suggested: several authors reported an altered
expression of
miRNAs in different cancers where they could function as both tumor
suppressor
or oncogenes. miRNA analysis has generally been performed by microarray
techniques in order to identify the specific expression profile
involved in
different types of cancers. On the basis on known complementary
nucleotide
sequences, computer-based prediction model have been developed in order
to
identify the specific target genes of miRNA; several relevant
association with
target genes, either oncogenes or tumor suppressors, have been found
but the
genetic pathways under miRNA regulation have still largely to be
determined The
laser microdissection is the method of choice to collect pure cell
population
starting from complex tissues. This technique has been already largely
used to
identify specific expression profile of mRNA in a single cell or in
different
cell compartment in the same tissue. In order to obtain in a series of
microdissected colorectal tumor tissue samples a sufficient quantity of
RNA for
gene expression analysis and for a specific related miRNA
quantification, we
have utilized several RNA extraction methods either specific for miRNA
(MIRACLE
miRNA Isolation kit, Stratagene) or for total RNA by a specific reagent
(Trizol, Invitrogen) or by a lysis buffer (Side Step Lysis and
Stabilisation
Buffer, Stratagene). We have performed a quantitative relative measure
of total
(18S) and miRNA by Real Time PCR (TaqMan); the miRNA were miR-31 and
miR-135-b.
These two miRNAs resulted altered in colorectal tumors in comparison to
relative normal tissues (Bandres et al, 2006). The relative
quantification is
obtained normalising to a RNU6b miRNA. All miRNA measurements were
performed by
TaqMan MicroRNA assay (Applied Biosystem). For the evaluation of total
RNA
extracted we have used a primers and probe for 18S RNA (Pre-Developed
TaqMan
Assay Reagents, Applied Biosystem). Results obtained by MIRACLE show
high
specificity in the miRNAs extraction and high accuracy for their
detection
starting from about 10 cells/tube. The other techniques (Trizol,Lysis
Buffer
Stratagene) were able to detect both total RNA and miRNAs with
sufficient
sensitivity, reproducibility and accuracy, even if the performances of
Stratagene buffer was affected, in same cases, by sample interferences
due to
the presence of DNA or proteins in the lysate. In all samples (n= 5),
miR-31
and miR-135-b resulted overexpressed in tumor microdissected samples in
comparison in normal paired tissue. P
004 MicroRNA
Expression Signatures as potential Biomarkers
in Thyroid Cancer. Astrid Potratz1,
Cara
Martin2,3, Simone Guenther1,O’Leary
J.23,
Orla Sheils2 (1)Applied
Biosystems, Applera
Deutschland GmbH, Frankfurter Strasse 129b, Email:
GuenthSM@eur.appliedbiosystems.com Cancer is
caused by abnormal
cellular proliferation and the inappropriate survival of damaged cells,
which
may result in tumor formation. Cells have developed several safeguards
to
ensure a correct and coordinated cell division, differentiation and
death. A defect
in the regulatory factors, e.g. tumour-suppressor genes and oncogenes,
will
increase the propability of tumour development. MicroRNAs
(miRNA), short non-coding,
single-stranded RNAs, constitute a novel class of negative gene
regulators.
Specific miRNAs may even control several target mRNAs resulting in
diverse and
complex biological processes. Recent evidence indicates that miRNAs
might also
function as physiological tumour suppressors and oncogenes. Repressing
the
expression of important cancer-related genes, miRNAs might therefore
prove a
valuable tool in the diagnosis and treatment of cancer. Thyroid
cancer cell line: The
expression profiles of 157 miRNAs in thyroid cancer cell lines have
been
analyzed using the Applied Biosystems TaqMan MicroRNA Assays. Specific
miRNA
patterns associated with different pathological pathways have been
identified,
indicating that miRNAs can be used to reliably discriminate between two
common
triggers of thyroid cancer. The predicted mRNA targets of these
differentially
expressed miRNAs belonged to genes involved in regulatory molecular
functions
such as signalling pathways, cell division control and transcription.
This
correlates nicely with the respective gene expression profiles using
the
Applied Biosystems Whole Genome Array System. There, genes involved in
the MAPK
signalling pathway, oncogenesis and transcriptional regulation where
shown to
be upregulated whereas several cell cycle regulators were found to be
down-regulated. Cervical
cancer cell line: The
expression profile of 180 miRNAs in two cervical cancer cell lines was
studied
using TaqMan miRNA Assays. miRNA was extracted using Ambions mirVana
miRNA
isolation system. miRNA extracted from histologically normal cervical
tissue
was used as a reference. A specific
different miRNA
expression signature in the cancer cell lines was observed compared to
normal
cervical tissue. Several of these differentially expressed miRNAs are
predicted
to interact with cell cycle regulatory molecules. These findings
highlight the
potential importance of miRNA molecules also in cervical cancer. The
determination of miRNA profiles
as a new class of biomarkers has the potential to significantly improve
diagnostic accuracy and prognostic information. P
005 Effects
of segmental trisomy on the expression of
microRNA genes by qRT PCR. Blatny R., Ivanek R.
& Forejt J. Institute of
Molecular Genetics AS
CR, Email:
blatny@biomed.cas.cz In our pilot
study, we have used
adult mice of the Ts43H mouse model of human aneuploidy syndromes
carrying
largest known segmental trisomy of an autosome with more than 300 genes
[1]. We
were interested in consequences of the segmental gene dosage imbalance
on
transcription of genes located in the trisomic region (up to 21 genes,
depending
on the tissue type) in comparison with genes located in the disomic
region (up
to 15 genes) of the same chromosome. MicroRNA
genes are known to be
example of non-codingRNA genes with strong regulatory potential and are
therefore candidate genes in study of development of different
pathological
states including aneuploidy syndromes. We have measured expression
levels of
three mature microRNA molecules located in a cluster inside of the
trisomic
region and one mature microRNA located on another chromosome. We have
selected
brain as our target tissue, since cognitive abilities were shown to be
affected
in the model [1], and we used liver as a control tissue. We are
reporting significant
differences in individual protein-coding genes between control animals
and
trisomic animals, mainly for genes in the trisomic region. The average
expression level of protein-coding genes in the trisomic region was
~1.6-fold
in both liver and brain, which corresponds with the altered gene dosage
and
does not indicate any kind of dosage compensation. In the disomic
region, the
average expression level was ~1.0-fold in liver and ~0.9-fold in brain,
which
indicates slight downregulation of the disomic part in brain. However,
statistical significance of this difference remains unclear. These
findings are
generally in agreement with studies on different mammalian models of
aneuploidy
and contrast with non-additive gene expression reported in some plants
and Drosophila
melanogaster . Finally, we
are presenting for the
first time measurements of gene-dosage effects on microRNA genes in a
mammalian
genome. The three microRNA genes located in the trisomic region were
upregulated ~1.5-fold. However, the microRNA gene located on another
chromosome
was found downregulated (0.8-fold). 1. Vacik, T.,
et al., Segmental
trisomy of chromosome 17: A mouse model of human aneuploidy syndromes. PNAS,
2005. 102(12): p. 4500-4505. P
006 MicroRNA
profiling of breast cancer using Locked
Nucleic Acid (LNA) based technologies. Jacobsen N.1,
Nørholm M.1,
Glue C.1, Stahlberg N.1, Eriksen J.2,
Svane I.
M.2, Flyger H.2, Balslev E.2,
Møller S.1,
and Litman T.1. 1Exiqon, Email:
jacobsen@exiqon.com Abnormal
expression of microRNAs
(miRNAs) in cancer implies that these small ~22-nucleotide molecules
play a
role in oncogenesis Therefore miRNAs may comprise a novel class of
diagnostic
and prognostic signatures. Here, we study the global expression
profiles of
miRNAs in breast cancer and normal adjacent tissue in order to identify
possible new biomarkers for breast cancer. Here, we
present miRNA expression
profiles from tumor and normal breast tissue, and found numerous
differentially
expressed miRNAs, including those previously reported to be associated
with
breast cancer, such as let-7a/d/f, miR-125a/b, miR-21, miR-32, and
miR-136. The
differential expression profiles from miRCURYTM LNA Arrays
have been
confirmed using real-time PCR assays. The real-time RT-PCR detection is
a
useful tool in addition to Northern blot analysis. We envision
that the different
platforms compared in the current study will facilitate an efficient
workflow
for identification of e.g. disease related miRNAs and subsequent
development of
reliable diagnostic and prognostic assays. P
007 Comparison
of miRNA expression patterns using the
total RNA extracted from formalin-fixed paraffin-embedded (FFPE) cells
and that
extracted from snap frozen. Li J.1,
Smyth P. 1,
Flavin R.1, Cahill S. 1, Denning K. 1,
Aherne
S. 1 Guenther S. 2, O’Leary J. 1,
Sheils O1 (1)Department
of Histopathology, Email:
GuenthSM@eur.appliedbiosystems.com Introduction: Archival
formalin-fixed paraffin-embedded
(FFPE) tissues represent an abundant source of clinical specimens;
however they
have limited utility in applications involving analysis of gene
expression due
to mRNA degradation and modification during fixation and processing.
Interestingly, miRNAs are a small class of RNA recently described as
playing
important roles in gene regulation, yet their robustness in FFPE is
largely
unknown. This study analyzed 160 miRNAs in paired snap frozen and FFPE
cells to
investigate if miRNAs may be successfully deteced in archival specimens. Methods: N-thy-ori
cells were grown to
confluence and aliquots with equal cell numbers were (a) snap frozen
and (b)
formalin fixed and paraffin embedded into a cell block. Total RNA was
extracted
using protocols: (a)Ambion mirVana miRNA Isolation kit for snap frozen
cells,
(b)Ambion RecoverAll Total Nucleic Acid Isolation Kit for FFPE cells.
The
quality and quantity of RNA yields was measured with Nanodrop and
TaqMan®
microRNA assays, Human Panel-Early Access Kit. Results: To achieve 50
ng of total RNA for
each RT reaction, 10,000 ng of total RNA (for 200 assays), was
extracted from
approximately 2x106 FFPE cells and 1.7x105 snap frozen cells.
TaqMan® analysis
showed a good correlation of miRNA expression pattern between FFPE and
snap
frozen cells, with R2>0.95. The mean of ΔCts (Cts_FFPE normalized to
Cts_snapfrozen) was -1.04107 and the median was -1.152 with
p<0.0001. 65.58%
of ΔΔCts (ΔCts normalized to ΔCts_mean), 101 out of 154 determined
assays, were
between +1 and -1. There was some outlying data in performing the
comparison
between snap frozen and FFPE cells, most notably miR-146 exhibited ~20
fold
decreased expression and miR-302b* ~8 fold increased expression. Conclusion: miRNA
extracted from FFPE blocks was
successfully amplified using Q-RT-PCR. The levels of expression of
miRNA
detected in total RNA extracted from FFPE were higher than that
extracted from
snap frozen cells when the amounts of total RNA were identical. It
seems
reasonable to conclude that this phenomenon was most likely caused by
methylol
cross-links between RNA and protein resulting small RNA molecules being
less
compromised than their larger counterparts. The majority of miRNAs
demonstrated
reliable expression levels in FFPE compared with snap frozen paired
samples
suggesting these molecules might prove to be robust targets amenable to
detection in archival material in the molecular pathology setting. P
008 A
new microfluidic Assay for the Analysis of small
RNAs. Marcus
Gassmann1 , Hans-Joachim Mollenkopf2,
Marc Valer3,
Martin Greiner1 1Agilent Technologies,
Waldbronn, Germany, 2Max-Planck
Institute for Infection Biology, Berlin, Germany and 3Agilent
Technologies Inc., Santa Clara, CA, USA Email:
marcus_gassmann@agilent.com MicroRNAs
(miRNAs) are short
20-22-nucleotide RNA molecules that have been identified recently as
sequence-specific regulators of many cellular processes such as
apoptosis,
proliferation and differentiation. Meanwhile hundreds of microRNAs have
been
discovered in the genomes of animals and plants, but they are only
beginning to
be classified by their functional roles. One of the major drawbacks is
the lack
of adequate analytical methods for the analysis of small RNA samples. Here we
describe a Lab-on-a-Chip
based assay that is able to perform very sensitive high resolution
analyses of
small RNA samples on the Agilent 2100 Bioanalyzer instrument. The assay
delivers information about integrity, size and concentration of small
RNA
species. Purified or enriched small RNA fractions, as well as total RNA
samples
in concentrations can be run in concentrations down to 100 pg/µl. The Agilent
2100 Bioanalyzer is a
microfluidic instrument platform designed for fast separation and
quantification of DNA, RNA, and proteins as well as flow cytometric
analysis on
cells. P
009 Multiplex
microRNA TaqMan® Low Density Array: A
High-Throughput Screening Tool for miRNA Profiling. Yulei Wang, Raymond
Samaha Applied Email:
wangyy@appliedbiosystems.com Main
Session: Single
Cell qPCR
Poster
number P 010 – P
011 Location:
Student Cafeteria P
010 Profiling
of the TrpC expression pattern in cerebellar
Purkinje cells by quantitative single-cell RT-PCR. Dragicevic E.1,Blum R.2,
Hartmann J.1, and Konnerth A.1 1Friedrich-Schiedel
Email:
elena.dragicevic@lrz.tu-muenchen.de The TRPC
(Transient Receptor
Potential Canonical) cation channel family consists of seven members;
TRPC1-7
(Clapham et al. 2001). TRPC subunits are expressed in many brain areas
but
little is known about their cellular function in neurons. It has been
reported
that the TRPC1 cation channel is involved in the mGluR1-dependent slow
excitatory postsynaptic current (ImGluR) in Purkinje cells (Kim et al.,
2003).
However, we found that ImGluR can be evoked in TRPC1 knock-out mice.
This
suggests a role for other TRPC family members in the activation of
ImGluR. To understand
the molecular basis of
TRPC-mediated postsynaptic currents, we investigated the cell
type-specific
gene expression patterns and levels of TRPC encoding transcripts in
single
Purkinje cells of the cerebellum using a quantitative Reverse
Transcriptase-PCR
(RT-PCR) approach (Durand et al. 2006). In our experiments, single
Purkinje
cell somata were obtained by whole soma suction from acute cerebellar
slices,
using microcapillaries. RT-reactions, followed by purification of
single cell
cDNA material were amplified in a real time PCR device (Lightcycler).
Quantification was performed by using high-resolution standard curves,
with
comparable efficiencies, for each subunit. In parallel, single cell
RT-reactions were validated by quantification of the house-keeping gene
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) in a second qPCR
reaction
using 1/10 amount of the single-cell cDNA. In addition, we use specific
TRPC
knock-out mutants to correlate the expression levels of TRPC subunits
with the
corresponding physiological phenotypes in Purkinje cells. Our findings
revealed that the
subunit TRPC3 (372 copies/cell) and not TRPC1 (44 copies/cell) is the
predominant TRP-family member in Purkinje cells of wild type mice.
TRPC4 (10
copies/cell), TRPC6 (4 copies/cell), and TRPC7 (5 copies/cell) have
been
detected in few copies, while TRPC5 was not detected yet. TRPC1
knock-out mice
show the same TRPC subunit expression pattern. The electrophysiological
analysis as well as the quantitative single cell RT-PCR experiments are
leading
to the conclusion that TRPC3 and not TRPC1 is the most likely molecular
candidate for ImGluR in cerebellar Purkinje cells of the mouse. Clapham, D.
E., Runnels, L. W.,
Strubing, C. (2001) The TRP ion channel family. Nat Rev Neurosci 387-96. Kim, S. J.,
Kim, Y. S., Yuan, J. P.,
Petralia, R. S., Worley, P. F., Linden, D. J. (2003) Activation of the
TRPC1
cation channel by metabotropic glutamate receptor mGluR1. Nature
426:285-291. Durand,
Marandi, Herberger, Blum
& Konnerth (2006) Pflügers Arch. Eur J. of Physiol. 451,
716-726. P
011 Changes
in the gene expression of mRNA transcripts for
insulin like growth factor (IGF-I), their receptor (IGF-IR) and
facilitative
glucose transporter (Glut-I) in IVM oocytes and preimplantation embryos
of S.C. Gupta, Neelam
Gupta,
Alok Pandey National
Bureau of Animal Genetic
Resources, Email:
guptasc@yahoo.com For low rate
of SCNT derived cloned
embryos reaching to normal and viable clones, one hypothesis is that
transcription of one or several developmentally important genes is
affected by
the in vitro environment possibly leading to the disturbance of process
of
differentiation and organogenesis. Therefore embryos exhibiting
abnormal
expression of embryonic genes may be an early indication of incomplete
reprogramming that could result in lower survival rates. The real-time
quantitative polymerase chain reaction (rtqPCR) has overcome the
limitations of
conventional, time consuming quantitative PCR strategies and has been
matured
into a routine tool to quantify gene expression levels, following
reverse
transcription (RT) of mRNA into complementary DNA (cDNA). It is a
sensitive and
very efficient technique to examine gene transcription patterns in
preimplantation embryos. One of the areas of interest in this regard,
is the
analysis of gene expression patterns in nuclear transfer (NT) embryos
to
dissect the processes that failed and develop means to overcome the
limitations
imposed by these factors. The main objective of this study was to
develop an
easy and rapid method for measuring gene expression in a single cell by
real-time PCR without RNA extraction and purification by using cell to
cDNA II
kit (Ambion). Based on the developed RT-PCR methodology, we constructed
cDNA
libraries with single matured oocytes at 0h, 6h, 12h, 18h and 24h of
maturation
and of embryos at different stages of development (2-cell, 4-cell,
8-cell,
16-cell, morula and blastocyst). Real time PCR cycler was performed
using
Brilliant SYBR Green Q-PCR master mix (Stratagene) to determine more
precisely
the transcription levels of IGF-I, IGF-II, and Glut-I in SCNT produced
buffalo
embryos derived from skin fibroblast cells at preimplantation
development. The
primers of selected genes GAPDH (reference) and IGF-I, IGF-II, and
Glut-I
(target genes) for expression studies were designed using the Gene Bank
sequences within only one exon in the range of 80-200 bp products.
Standard
curve for reference and all the target genes were developed for the
relative
quantification of gene expression. IGF-I transcript expression was
clearly
visible only in matured oocytes. It was elevated up through 12h of the
culture
and then declined gradually at the end of 24hrs maturation. IGF-IR, was
expressed throughout preimplantation development upto the blastocyst
stage,
while the expression of facilitative glucose transporter (Glut-I) was
also
expressed in all stages studied. Gene expression analysis of those
genes which
plays a important role in activation and reprogramming of matured
oocytes and
cloned embryos would enable us to better understand the early
biological events
during preimplantation after nuclear transfer. Session:
Immuno - qPCR
Poster
number P 012 – P
013 Location:
Student Cafeteria P
012 Real-time
immuno-PCR: a new perspective for prion
blood screening tests. Ruelle V., ElMoualij
B.,
Heinen E. and Zorzi W. Center of
research on Prion Proteins, Email:
v.ruelle@ulg.ac.be Prion
diseases such as
Creutzfeldt-Jakob of human, scrapie of sheep and bovine spongiform
encephalopathy of cattle are fatal neurodegenerative disorders
characterized by
behavioural and locomotor changes, cerebral amyloid plaques and
spongiform
degeneration of the brain1. In 1996, the first case of
variant of
Creutzfeldt-Jakob disease (vCJD) was diagnosed in the The classical
techniques used to
diagnosed prion protein (Western blot assays and enzyme-linked
immunosorbent
assays)5 are not sufficiently sensitive to detect the low
levels of
prion in the blood. Consequently, it is necessary to develop a
sensitive
technique allowing the diagnosis of prion protein in blood. In this
study, we therefore focussed
on the detection of the prion protein in blood by
immuno-quantitative-PCR
(iqPCR)6. This technique combines the sensitivity of PCR, by
an
exponential amplification of reporter DNA, and the specificity of the
detection
of antigens, by antibodies in an ELISA format7-8. To
illustrate the
advantages of iqPCR, we have compared it with a conventional ELISA
technique in
experiments aimed at detecting the resistant form of prion protein in
human
plasma. Using iqPCR, a minute quantity of prion was detected in blood
spiked
with infected CJD sample with a detection threshold at least 400-fold
lower
than classical ELISA. The iqPCR
technique being
ultra-sensitive, it could be a technique of choice for the development
a new
blood screening tests allowing the prion protein diagnosis in infected
human
and animals ; both at ante-mortem and post-mortem stage. P
013 Andreas
Fischer1, Thorsten Kuczius2,
Christof von Eiff1,
Georg Peters1, Karsten Becker1 Email:
a.fischer@uni-muenster.de TSS-mediating
staphylococcal toxins:
A novel quantitative real-time immuno-PCR approach for ultra-sensitive
detection of SEB and TSST-1 Staphylococcus
aureus is a major
human pathogen characterized by a strain-dependent spectrum of
virulence
factors. One of these is the family of the bacterial pyrogenic toxin
superantigens (PTSAg), comprising the toxic shock syndrome (TSS)
mediating
toxins TSST 1 and staphylococcal enterotoxin B (SEB). As morbidity and
mortality from TSS are substantial, early and reliable recognition of
TSS is critical.
In addition to their nature as superantigens, enterotoxins also
function as
potent gastrointestinal toxins with a major public health impact. Immunological
methods used until now
are known to be limited in sensitivity and specificity, revealing an
obvious
need for new highly sensitive and specific methods for the detection of
staphylococcal toxins. For this reason, two quantitative real time
immuno-PCR
(qRT-iPCR) approaches for the detection of SEB and TSST-1 have been
developed. The detection
of TSST-1 and SEB,
respectively, was achieved by coating toxin-specific polyclonal sheep
antibodies (ABs) to microtiter plates in order to capture the target
superantigens followed by specific detection of the antigen-AB complex.
The
resulting immunocomplex was subsequently detected using a covalent
antibody-DNA
complex, which was synthesized using amino-modified and
maleimide-activated
reporter DNA and N-succinimidyl-S-actyl-thioacetate-modified secondary
detection antibodies. Quantitative real-time amplification of the
reporter-DNA
was performed for final detection of the toxins. By usage of this qRT-iPCR technique, superantigen toxin was highly reproducible detected at approximately 10 to 100 pg/ml (0,4 to 4 amol/µl), thereby lowering the limit of detection (LOD) of these toxins by a factor of up to 100 compared to commercially available EIAs. Furthermore, qRT-iPCR offers a high versatility, giving the opportunity of adapting the protocol to detect a broad range of antigens, provided that antibodies for the desired antigens are available. Covalent attachment of different reporter-DNAs to specific antibodies could be used to extend the qRT-iPCR to a multiplex detection platform. Session:
Pre-analytical-Steps
Poster
number P 014 – P
022 Location:
Student Cafeteria P
014 Evaluation
of different RNA extraction methods for
small quantities tissue from the marine flatworm Macrostomum lignano. Plusquin M., Smeets K.,
Geerdens E., Remans T.,
Cuypers A., Artois T. Email:
michelle.plusquin@uhasselt.be Highly
sensitive techniques for
transciptome analysis, such as real-time PCR, microarrays and others
currently
used in functional genomics require a high integrity and quality of the
RNA, as
well as reproducibility between replicates of the same tissue. Our
test-organism Macrostomum lignano is a small marine flatworm
that has an
average weight of 350 µg. Because culture of Macrostomum
lignano is
labour intensive our goal was to isolate RNA from small quantities of
sample
material. Samples were lysed with different mechanical lyses such as
pulverisation, mixing with steal beads and sonication. Total RNA was
then
extracted using TRI- Reagent ®, as well as commercial kits based on
RNA binding
to silicon membranes (Qiagen, Roche) or magnetic beads (Invitrogen).
RNA
concentrations and purity was assessed using a nanodrop ® -ND 1000
UV-Vis
Spectrophotometer using a 1 µl aliquot of the total RNA
solutions. RNA
integrity of the extracted RNA molecules was evaluated in 1 µl
using an Agilent
2100 Bioanalyzer with the RNA 6000 Pico labChip® kit. P
015 Automated
RNA extraction using new Agencourt SPRI
technology. Souquet M. Email:
msouquet@beckman.com Agencourt
RNAdvance is an Agencourt
SPRI® paramagnetic bead-based purification system for the isolation
and
purification of total RNA from cultured eukaryotic cells, tissue or
blood.
Agencourt RNAdvance is a consistent and automation-friendly method for
utilization in downstream microarray and real-time gene expression
analysis.
This technique reliably delivers high recovery and purity without the
need for
filtration or centrifugation and is especially well suited for
automation using
Beckman Coulter Biomek® solutions. P
016 Advanced
Data Mining Software for Applied Biosystems
RT-PCR Data Analysis. de Alarcon P.1 Ferlinz A.2 1Integromics
SL, Email:
pedro.dealarcon@integromics.com Great
advances in instrumentation,
accurate fluorescence detection, improvements in reagents (eg. by means
of ABI
Taqman probes) and protocols have increased the use as well as the
range of
applications of quantitative RT-PCR experiments. Nowadays, RT-PCR is
the best
technique for relative gene expression quantification and it is
expanding to
other areas like miRNA profiling, biomarker discovery, diagnostics and
Copy
Number Analysis. Moreover, the amount of data generated is also growing
as
experiments are being performed within a high-throughput context. In
this
sense, advanced bioinformatic tools that help researchers in the
analysis and
interpration of raw data are demanded by the scientific community as a
key
complement to the instrumentation and reagents. In the present poster,
Integromics introduces a new high-performance software specifically
designed
for the new era of quantitative RT-PCR. The software has been built
around four
principles, namely: high-throughput statistics and data mining,
interactive
visualization, functional interpretation and extensible modular
approach.
State-of-the-art statistics are key to provide quality control and
analysis of
raw data for filtering of outliers and noisy experiments. Further
procedures
are provided towards the assesment of expression stability of
endogenous genes
(like Normfinder), differential expression by means of statistical
tests, and
clustering techniques for grouping similar expression profiles. All the
statistics are implemented in the R language which ensures the
capability of
dealing with large amounts of data as well as benefiting from the
existing
libraries in Bioconductor (www.bioconductor.org). The software is
implemented
as a plugin of Spotfire Decisionsite, a leading application for
professional
and interactive visualization of multidimensional data. Visualizations
in
Spotfire facilitate the task of exploring large datasets (possibly with
hundreds of genes and samples) so that it becomes easier to pose
interactive
queries and focus on relevant results. Even more, once the results from
the
analysis highlight the existence of characteristic expression profiles,
it is
crucial to enrich the numerical information with functional annotations
provided by content repositories like the Panther database
(www.pantherdb.org).
As mentioned above, RT-PCR is a powerful technique that can be applied
in many
different contexts like relative quantification, miRNA profiling or
genotyping.
However, it is unrealistic to provide software that cannot be adapted
to such
diversity. In this sense, the software implements analytical workflows
that can
be easily adapted to specific user needs or experimental settings. A
workflow
is a set of sequential steps that guides the user from the importing of
raw
data to the final analysis in a very natural manner. This facilitates
the use
of the tool by non-experts in biostatistics but does not limit its
power as
statiticians can add or manipulate the procedures contained in the
software. P
017 Preamplification
of Sample Limited Specimens for
Real-Time Gene Expression Analysis. Junko Stevens 1,
Renata Coudry2, Cynthia Spittle3 1Applied Email:
stevenjn@appliedbiosystems.com Accurate gene
expression profiling
can be compromised by the quantity of RNA that is isolated from cells
or
tissues. We have developed a robust solution for uniform amplification
of cDNA
prior to quantitative, real-time PCR. TaqMan® Preamp Master Mix
allows
preamplification of up to 100 gene targets simultaneously using the
TaqMan®
Gene Expression Assays as the source of pooled gene-specific primers.
TaqMan
assay-based preamplification preserves equilibrium of targets and
retains the
relative copy numbers of starting targets in a reproducible and precise
manner.
Preamplification of random-primed cDNA is independent of amplicon
distance from
the 3’ end, making it amenable to use with partially degraded or viral
RNA.
Uniformity of preamplification was demonstrated using Laser Capture
Micro
dissection (LCM) in formalin-fixed, paraffin embedded (FFPE) tissue.
RNA
extracted from clinical samples was reverse transcribed to cDNA using
High
Capacity cDNA Reverse Transcription Kit The simple workflow enables
researchers
to enrich the amount of limited RNA samples uniformly within 1.5 hours. P
018 qPCR
with mRNA isolated from urothelial cells from
urine. Bontrup H., Delbanco
J.,
Bruening T., Johnen G. BGFA, Email:
bontrup@bgfa.ruhr-uni-bochum.de Objectives Bladder
cancer in chemical workers
is an occupational disease associated with previous exposure to
aromatic
amines. Currently, urine-based markers used for screening of high-risk
collectives are not of high sensitivity. To detect cancer at earlier
stages
more suitable non-invasive markers are necessary. Some promising new
tumor
markers are based on mRNA quantitation. The aim of this study was to
establish
and optimize a practical and efficient mRNA isolation method that
allows
applying qPCR-based assays with urothelial cells from urine. Methods Six different
isolation methods on the basis of commercially available kits were
compared
using urine samples of healthy donors and a control RNA with known
concentration. A situation was simulated comparable to sample
collection in a
clinical setting. Cells were collected from urine by centrifugation and
transferred to a buffer according to the manufactures recommendations.
After
short (48 h) storage at -20°C the mRNA isolation was performed. In
all tested
assays mechanical disruption of the cells was identical. The six
isolation
methods differed by DNA removal step (DNase treatment or special DNA
column)
and material of the RNA columns. After isolation, extracted RNA was
transcribed
to cDNA and quantified on a LightCycler system using an adapted
Taqman-based
assay for ß-Actin (FDI, Results It was
possible
to establish a system for mRNA isolation from urine but the analysis
showed
that urine as a starting material yields high contaminations with
genomic DNA.
Therefore, DNA removal is essential to obtain mRNA of sufficient
quality for
qPCR. Our results show that DNA could not be removed adequately by
DNase
digestion. Better yield and purity was only possible with application
of a
special glass fibre column that selectively binds DNA. This also allows
to
recover the DNA and use it for other applications. Conclusions Most of the
six
tested methods for mRNA isolation from urine are generally suitable for
downstream qPCR applications. However, best results can be obtained
with a DNA
column-based method (INVITEK, This study
was in part supported by
Fujirebio Diagnostics Inc. P
019 Quantitative
mRNA expression of the embryonic stem
cell marker POU2 in early developmental stages of Kaja Helvik
Skjærven, Elisabeth
Holen National Email:
ksk@nifes.no Embryonic
stem cells (ESC) from
Atlantic cod (Gadhus morhua) have been isolated and cultured
successfully.
The cultured cells showed characteristic features for ESCs, including
spontaneous differentiation and the ability to form embryoid bodies
following
retinoic acid treatment. In addition the ESCs could be directed to
differentiate into neuronal like cells upon stimulation. Detection of
genes that identify
ESCs from other differentiated cells is vital for the method. POU2 is
one such
gene, known to regulate potency, and self renewal in ESCs. Class V
POU5f1 genes
are found in blastula cells of different species; Oct4 in mammals and
POU2 in
zebrafish. We have previously described a fragment of POU2 isolated
from
Atlantic cod that is 100% identical to zebrafish POU2 at the amino acid
level,
but not at the nucleotide level. Using real-time PCR technique, we
report that
blastula cells taken from 1 and 1.5 days post fertilization (DPF)
express the
highest transcriptional levels of POU2. At the early gastrula stage (2
DPF) a
1.5 fold reduction in POU2 expression was correlated to the presence of
some
early differentiating cells. By the late gastrula stage (3 DPF), POU2
expression was barely detectable, and coincided with the presence of
many
differentiated cells. We conclude that POU2 can be used as an ESC
marker for
Atlantic cod, and the method represents an important new research tool
for use
in marine teleost species. P
020 Relative
mRNA quantification models and the impact of
RNA integrity. Simone Fleige and Michael
W.Pfaffl Physiology
Weihenstephan, Email:
fleige@wzw.tum.de Quantitative
real-time PCR has
become one of the most widely used methods of gene quantitation. The
method
exhibits a large dynamic range, boasts tremendous sensitivity, has
little to no
post-amplification processing, and is amenable to increasing sample
throughput.
An essential requirement for a successful quantitative mRNA analytics
using
qRT-PCR is the usage of intact RNA. Low-quality RNA may compromise the
derived
expression results. The importance of RNA quality for the qRT-PCR was
analyzed
by determining the RNA quality of different bovine tissues and cell
lines
(n=11). Diverse artificial and standardized RNA degradation levels were
assessed using the capillary electrophoresis system (Agilent
Bioanalyzer 2100).
The RNA quality was classified according the RNA integrity number
(RIN).
Further on, a research into the effect of different length of amplified
products and RNA integrity on expression analyses was investigated.
Degraded
RNA interferes with qRT-PCR performance as such, expressed as Ct value,
whereas
PCR efficiency is minor effected by RNA integrity. Statements about
importance
of normalization could be confirmed by our investigations, consequently
we
commended an efficiency-corrected relative quantification strategy and
normalization with at least one internal reference gene. We can
recommend a RIN
higher than five and a PCR product length up to 200 bp as a minimal
requirement
for a successful and reliable quantitative real-time RT-PCR
quantification. P
021 REPEATABILITY
OF HOUSE DUST SAMPLES IN RELATION TO
QUANTITATIVE PCR OF MICROBES. Pasi Kaarakainen1, Asko
Vepsäläinen1,
Aino Nevalainen1, Teija Meklin1,2 1National
Public Health Institute,
Department of Environmental Health, Kuopio, Finland and 2Technology
Centre Teknia Ltd, Kuopio, Finland Email:
pasi.kaarakainen@ktl.fi Possible
specific associations
between fungal species in our indoor environment and adverse health
effects,
such as respiratory symptoms, asthma and allergy are a major topic of
interest.
House dust sampling has been used to assess microbial exposures and to
describe
microbial populations in indoor environments. However, the
repeatability or
representativeness of such sampling has not been thoroughly validated. In this
study, the repeatability of
house dust sampling was tested. Furthermore, the seasonal variation of
the
microbial populations was analyzed to assess the representativeness of
a single
sampling. QPCR was used for the identification of 16 species or assay
groups of
fungi from house dust samples from five houses without any moisture
damage. The
repeatability of the analysis of a floor dust sample with qPCR was
tested by
isolating DNA from homogenized dust samples as five parallel samples.
Collecting samples in four different seasons enabled the consideration
of
seasonal variation. The highest
concentrations from
analysed species or assay groups of fungi were observed for Aureobasidium
pullulans, Penicillium/Aspergillus/Paecilomyces variotii group, Aspergillus
penicillioides, Cladosporium herbarum and Cladosporium
cladosporioides, respectively.
Fungal concentrations varied between seasons depending on analysed
species or
assay groups. Concentrations of A. pullulans and two Cladosporium
species
were at their highest in summer and autumn while for Penicillium and
Aspergillus species, the differences between different
seasons were not
so obvious. The
repeatability of the parallel isolations of DNA was good for most of
the
analysed species. The best repeatability was observed for assays of C.
herbarum and P. chrysogenum with ICC value (similarity of
the
parallel samples) 84.5 and 83.1 %, respectively. ICC was over 70 % also
for six
and over 60 % for three other assays. In summary, our results suggested
that qPCR
is a promising tool for the microbial analyses from house dust samples
in
epidemiological studies. In determining the qualitative procedures of
the
method and the reliability of the results, homogenization of the sample
matrix
and the number of the repeats of analyses are the primary issues. P
022 RNA
Integrity database: A web repository enabling RNA
trace comparisons. Hans Brunnert1,
Martin
Greiner1, Marcus Gasmann1, Michael Kim2,
Marc Valer2 1Agilent
Technologies, Email:
martin_greiner@agilent.com Lab-on-a-Chip
devices are broadly
used for RNA integrity analysis on Micro array and qPCR gene expression
analysis. This creates the need and opportunity for users to screen and
validate
RNA traces for relevance and troubleshooting. Here we describe the
design of
the backbone as well as examples of use for a new RNA profile web
database. The
database aims to host a large variety of sample types spanning
different genus,
tissues and sample treatments, although the database is initially
limited to
contain bioanalyzer traces. Each electropherogram is annotated with
sample
source details as well as analytical data like UV, Ribosomal ratios,
RIN and
more. They also include details on the RNA extraction and the
downstream
experiment. By design the database is open to the scientific community: free querying and curated contributions for individuals as well as large batch uploads from core labs. Individuals are able to compare their own results with those of others with similar samples and protocols. It also allows comparison of alternative RNA isolation methods based on its resulting electrophoretic traces. Session:
qPCR
BioStatistics & BioInformatics
Poster
number P 023 – P
028 Location:
Student Cafeteria P
023 GenEx
for Real-time PCR Gene Expression Profiling. Forootan A., Kubista M.,
Sjögreen B and Andrade J
M TATAA
Biocenter, Sweden MultiD
Analyses, Sweden Dept of Analytical Chemistry, University of A Coruna
Center
for Applied Scientific Computing, Lawrence Livermore National Laboratory Email:
amin.forootan@multid.se The
extraordinary sensitivity and
virtually unlimited dynamic range of real-time PCR makes it the
preferred
technology for gene expression profiling. Candidate marker genes are
identified
by microarray technology and validated on representative samples by
real-time
PCR. False leads are discarded, resulting in very powerful panels of
expression
marker. Such panels can be developed for staging of disease,
classification of
cells, studies of expression pathways, effects of drugs and the like.
The
recent development of high throughput real-time PCR platforms such as
the 384
well plate instruments from Applied Biosystems and Roche, and most
recently the
48x48 (2304 wells) microfluidic card from Fluidigm will spur the
development
further. To extract maximum information from expression profiling
experiments
powerful statistical and mathematical methods are needed to find
correlations
between the measured profiles and identify the genes and samples that
show
common behavior. Here we present GenEx, which is the first windows
based
package dedicated for real-time PCR expression profiling. GenEx has
user-friendly interface that makes advanced analyses really easy. Data
from
multiplate experiments are readily pre-processed and analyzed by
methods such
as geNorm , Normfinder, Principal Component Analysis, Hierarchical
clustering,
Self-Organizing Maps, Potential curves and much more. The results are
presented
in highly attractive plots. www.multid.se The Real-Time
Polymerase Chain
Reaction, M. Kubista, J.M. Andrade, M. Bengtsson, A. Forootan, J.
Jonak, K.
Lind, R. Sindelka, R. Sjöback, B. Sjögreen, L. Strömbom,
A. Ståhlberg, N.
Zoric, Molecular Aspects of Medicine (2006) 27, 95-125 P
024 EM
algorithm for gene copy number estimation using
TaqMan® assays. Catalin
Barbacioru, Kelly Li,
Raymond Samaha and Katherine Lazaruk Applied
Biosystems, Email:
catalin@appliedbiosystems.com Genetic
variation in the human
genome takes many forms, ranging from large, microscopically visible
chromosome
anomalies to single-nucleotide changes. Deletions, insertions,
duplications and
complex multi-site variants, collectively termed copy number variations
(CNVs)
or copy number polymorphisms (CNPs), are found in all humans and other
mammals.
CNVs influence gene expression, phenotypic variation and adaptation by
disrupting genes and altering gene dosage, causing diseases, as in
microdeletion or microduplication disorders, or confer risk to complex
disease
traits such as HIV-1 infection and glomerulonephritis. Recently,
TaqMan® gene
copy number assays have been developed for detection of genetic
variation at
gene level using primers and probes designed for genomic DNA sequences.
Each
well is duplexed with two assays. The FAM™ dye-based assay
is
designed to detect the genes-of-interest and the VIC®
dye-based
assay for detection of the reference gene, RNase P. The difference
between FAM
and VIC measurements (dCT) is indicative of the relative abundance of
the
gene-of-interest against 2 copies per diploid genome regardless of the
status
of the gene-of-interest. In this study, we present an algorithm for
gene copy
number estimation from TaqMan® gene copy number assays
based on EM
algorithm for mixtures of normal distributions. After removing
technical
outliers from data, the algorithm finds maximum likelihood estimates of
parameters in probabilistic models, where the model depends on
unobserved
samples copy number of the gene-of-interest. Estimates of the gene copy
number
and confidence levels in predicted copy numbers are reported for each
sample.
Under current protocols, we are capable of distinguishing up to 8
copies of the
gene of interest with at least 95% confidence, assuming 100% efficiency
of the
FAM™ dye-based assay. To evaluate
this algorithm, we
present experimental results for 5 important drug metabolism genes
(CYP2D6,
CYP2E1, CYP2A6, GSTM1 and GSTT1) on 270 individual samples from
International
HAPMAP Project representing 4 different populations (Caucasian,
African,
Chinese and Japanese). Copy number analysis for these genes shows
perfect
consistency for sample duplicates. Copy number variation (from 0 to 4)
is
observed for all 5 genes. Significant differences of copy number
frequency in
these populations are revealed for CYP2A6, CYP2D6, GSTM1 and GSTT1.
Therefore,
data presented here is most relevant for highlighting variable regions
of the
genome that warrant consideration in disease studies. Furthermore,
combining
this data with SNP data for the same genes, we demonstrate that
departures from
diploidy can cause apparent genotyping failure and give inaccurate
genotyping.
Therefore, measuring copy number variation in these genes is an
important
complement to genotyping assays. P
025 Ross Saad1,
David Chiew1,
Matthew Herrmann1, Valin Reja1, Michael W.
Pfaffl2 1Corbett
Research, Mortlake, NSW, Email:
michael.pfaffl@wzw.tum.de REST 2007 is
new standalone software
for analyzing gene expression using real-time amplification data. The
software
addresses issues surrounding the measurement of uncertainty in
expression
ratios by introducing randomization and bootstrapping techniques. New
confidence intervals for expression levels also allow measurement of
not only
the statistical significance of deviations but also their likely
magnitude,
even in the presence of outliers. Whisker box-plots provide a visual
representation of variation for each gene, highlighting potential
issues such
as distribution skew. REST 2007 builds on its predecessor REST 2005
with
significant improvements to randomization algorithms. This new revision
introduces alternative data inputs such as single sample efficiency and
amplification take-off point, alleviating the need to set amplification
plot
thresholds. http://REST.gene-quantification.info/ P
026 Using
fuzzy logic algorithm and gene expression
database ONCOMINE in COPD outcome forecasting. Shilov B.V., Bukreeva
E.B. Email:
shilov@ssmu.ru Chronic
obstructive pulmonary
disease (COPD) occupies one of the first places in frame of a sickness
rate in
the world. COPD is a focus of chronic inflammation in organism. Chronic
inflammation and its local repeated stress have long been known to be
risk
factors for cancer. Moreover risk of carcinogenesis increases under
influence
infectious pathogen. Examples include: Helicobacter pylor bacterial
infection
and gastric adenocarcinoma; hepatitis B virus and hepato-cellular
carcinoma;
chronic bowel disease and colon carcinoma; EBV and nasopharyngeal
carcinoma in
humans. Thus risk of malignant outcome of disease can be enlarged if a
patient
with COPD has colonization of the infectious agents in his organism. Infectious
agent of inflammation
influences on squamous cell methaplasia development in COPD patients.
This fact
accompanies by increase of squamous cell quantity and rising in
brush-biopsies
reserved cell number belong in G2 phase of cell cycle. Our
investigation
included survey of 37 patients with COPD infectious exacerbation. In
conditions
of similar morphological and pathological changes different patients
had
various genes expression level. There is
known differences in these
genes expression beside healthy humans and malignant disease patients.
Dates
about these differences were obtained from ONCOMINE database with free
access.
ONCOMINE, a cancer microarray database and web-based data-mining
platform aimed
at facilitating discovery from genome-wide expression analyses for
elucidation
state of indicated genes expression in patients with squamous cell
methaplasia.
Search and analysis ONCOMINE results we used in fuzzy logic algorithm
for
expert fuzzy rules forming. When dealing
with gene expression
data, the problem is even more complicated, because no expert exists to
determine what defines a “normal” expression level. Using fuzzy logic,
the full
range of expression data is first measured and is then broken into
discrete
subsections based on the observed data. These discrete subsections then
provide
a qualitative description of the data. Use the
algorithm has allowed
patients classifying on the gene expression basis in groups with
different
forecast of the upshot of the disease. Three groups were chosen:
favorable
forecast (5 patients), stabile condition of the chronic inflammation
(24
patients) and group with high probability squamous cell methaplasia
development
(8 patients). P
027 qRT-PCR
applied to the comparative analysis of plant
splice variants in time-course water-stress experiments. Cantale C., Latini A.,
Sperandei M. and Galeffi
P. BAS BIOTEC GEN,
ENEA, Italy Email:
cantale@casaccia.enea.it The
understanding of the plant gene
response to the environmental stresses at the transcriptional level is
a main
challenge for dissecting their role in the stress tolerance. We
analysed the
expression profile of a DREB-homologous gene, TdDRF1, using Real Time
RT-PCR in
various different Triticum durum and Triticale cultivars
upon
dehydration, both in greenhouse and in controlled field experiments.
The TdDRF1 gene is alternatively spliced and results in three
splice
variants, two of
them codifying for putative transcriptional activators and the third
one
producing an abortive putative protein with an unknown function. For
each of
the examined cultivars, a time-course experiment under water-stress is
carried
out including two groups, the control plants, that continue to be
watered and
the stressed plants, that are no more watered. The experiments are
carried out
using plants at the same growth level and some leaves are collected and
pooled
at fixed times to average the possible individual and position/soil
variability. The three
splice variants, TdDRF1.1, TdDRF1.2 and TdDRF1.3 ,
are 1455bp, 1367bp and
1314bp
respectively and a high sequence identity among the exons in the
regions
flanking the splicing recognition site prevents the design of primers
able to
select exclusively each transcript, as demonstrated by the
co-amplification
observed in experiments using SYBR Green technology. Thus, the qRT-PCR
experiments have been carried out using the TaqMan probe-based
technology and
the 18S rRNA was used as an internal control. It has to be
underlined
that we were interested in comparing the three transcripts in the same
samples
of interest. As the expression levels of the three target genes are
largely
different and one is present in very small amounts, the experimental
conditions
resulted suboptimal in some cases, as demonstrated by the amplification
plots. Amplification
efficiencies of each
sample have been evaluated from raw data using LinReg software and show
significant differences among the targets. The amplification efficiency
of each
transcript has been estimated also by linear regression using serial
dilution.
We report our results obtained using two different methods, Q-gene and
a
combination of LinReg software and GED formula. The last methodology
has been
applied using both individual and averaged amplification efficiencies.
Furthermore, the application of the GED formula to our data results in
three
possible data combinations, that are compared and discussed. P
028 Simplified
simulation model of polymerase chain
reaction (PCR). Anneliese
Ernst1, Veronique
Creach2, Sven Becker3, Lucas J.Stal4,
Peter
M.J. Herman4. 1Fraunhofer
IGB, Email:
ern@igb.fhg.de A simplified
model of polymerase
chain reaction (PCR) is capable to describe essential characteristics
of the
amplification kinetics of this cyclic reaction. The model is based on
the
assumption that the PCR is designed and conducted in such a way that
(one) PCR
primer(s) limit(s) the formation of double-stranded PCR products. The
absence
of other limiting factors such as nucleotide limitation, enzyme
limitation or
shortage of the fluorescent dye SYBR Green® greatly reduces the
number of
parameters required to simulate real-time kinetics of individual
amplification
reactions in more explicit models. With a self-developed FORTRAN code,
we can
fit experimental data using the initial concentration of the limiting
PCR
primer to calibrate the fluorescence emitted by Sybr Green. This fit
provides a
novel method to estimate the initial concentration of target DNAs in a
PCR
sample. The method does not rely on external or internal standards. The
simulations provide explanations for the occurrence of PCR biases and
artifacts; and they are a valuable tool for teaching purposes, and in
optimizing PCR protocols. Session:
New Diagnostic
applications with qPCR
Poster
number P 029 – P
065 Location:
Student Cafeteria P
029 Application
of qRT-PCR in reproductive toxicology. Wolfgang R.
Schäfer, Wolfgang R.
Deppert, Michael Simon, Katrin Roth, Aida Hanjalic-Beck, Claudia
Nöthling,
Hedwig Austermann-Hesse, Hans Peter Zahradnik Universitäts-Frauenklinik
Freiburg, Germany Email:
wolfgang.schaefer@uniklinik-freiburg.de Drugs and
xenobiotics are known to
induce changes in gene expression which are often more sensitive and
characteristic than currently employed toxicological endpoints. We
describe the
use of quantitative real-time RT-PCR in the development of an in
vitro -test
to assess possible hazards of chemicals on endometrial functions
essential for
embryo implantation. Our work is part of the integrated project ReProTect
(EU)
developing new -in vitro_-tests which are required under the new
European
chemicals legislation (REACH) to reduce, refine or replace the use of
laboratory animals. Within the ReProTect research area of
“Implantation”
we are exploring on the mRNA level predictive toxicological endpoints
in human
endometrial explants (e.g. leukaemia inhibitory factor, LIF;
progesterone receptor;
estrogen receptor α; calcitonin; cyclooxygenase-2; corticotrophin
releasing
hormone receptor 1; VEGF-receptor 2, KDR). The goal of
the human endometrial
explants assay is to detect chemical effects on endometrial functions
which are
essential for embryo implantation. This test is a closer approach to in
vivo -conditions than primary or permanent cell cultures.
Endometrial tissues
from the proliferative and secretory phase of the menstrual cycle are
obtained
by aspiration curettage from premenopausal women. The endometrial
biopsies are
chopped into pieces of 1-2 mm/side and cultured in sex steroid
supplemented
medium without phenol red in a humidified atmosphere at 37 °C in 5
%
CO<sub>2 for 6-24 hrs. Test chemicals are administered during
this incubation
period and compared to negative controls with vehicle alone. Facing the
low tissue amounts of
human endometrial explants which are available for each incubation
(approximately 20 mg) quantitative real-time RT-PCR (LightCycler 480,
Roche) is
applied as the major analytical method. In order to identify predictive
toxicological endpoints among a larger number of target genes assays
from the
highly versatile Universal Probe Library (UPL; Roche) are used.
Evaluation is
performed by calibrator-normalized relative quantification. A status
report of
our project will be presented. It includes calibrator standard curves
for
efficiency correction, selection of housekeeping genes and preliminary
data of in
vitro -testing with the antiprogestin reference compound RU 486. This project
is funded by the European Commission ( ReProTect , Project
no.: LSHB-CT-2004-503257) P 030 Nataša Toplak1, Miha Kovač2,
Minka Kovač1, Tjaša Cerar3 and Eva Ružić Sabljić3 1Omega d.o.o.,
Dolinškova 8, 1000
Ljubljana, 2Poljane Grammar School Gimnazija Poljane,
Strossmayerjeva 1, 1000 Ljubljana and 3University of
Ljubljana,
Faculty of Medicine, Institute of Microbiology and Immunology, Zaloška
4, 1000
Ljubljana Email:
omega@omega.si Worldwide, many infections caused by viruses, bacteria, or parasites are known to be tick-transmitted zoonoses. Tick-borne diseases are a major threat to mammals and humans during outdoor activities in spring and fall. One of the most important tick-borne diseases is Lyme borreliosis caused by spirochetes within the Borrelia burgdorferi sensu lato complex. The different species of this group are involved in clinical manifestat |