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qPCR 2007 Symposium TALK
Presentations
Thomas W.
Myers Program in
Core Research, Roche
Molecular Systems, Email:
thomas.myers@roche.com Main Session:
microRNA
– siRNA Applications
Chair: V. Benes / G.
Shipley Lecture hall HS 14 Meister G. Max Planck
Institute of
Biochemistry, Email:
meister@biochem.mpg.de Small
regulatory RNAs such as short
interfering RNAs (siRNAs) or microRNAs (miRNAs) have been discovered in
the
past and it is becoming more and more apparent that these small
molecules have
key-regulatory functions. Such small RNAs are found in all higher
eukaryotes
and play important roles in cellular processes as diverse as cell
differentiation, hormone secretion or stress response. SiRNAs guide
sequence-specific cleavage of perfectly complementary target RNAs,
whereas
miRNAs associate with partially complementary target mRNAs and repress
their
translation. It is becoming more and more apparent that miRNAs can also
influence the stability of mRNAs by guiding de-adenylation followed by
mRNA
decay. Interestingly, miRNA expression has also been associated with
different
diseases including neurodegenerative disorders as well as cancer. A
detailed
investigation and an accurate quantification of miRNAs in different
tissues
will therefore provide a better understanding of the molecular basis of
such
diseases and may ultimately lead to novel means of diagnosis. MicroRNA
profiling toolbox: points to consider. Vladimir Benes, Mirco
Castoldi, Sabine Schmidt, Martina Muckenthaler EMBL,
EMBL-University of Email:
benes@embl.de MicroRNAs
(miRNAs) are now
recognized as an important class of small RNA molecules that play an
important
role in the regulation of gene expression. Research into non-coding
RNAs in
particular miRNAs is currently one of the hottest yet challenging areas
of
molecular biology, in part due to the complex biogenesis of this class
of
molecules. Two main properties of these molecules, namely their length:
being
only ~22 nucleotides long and the high frequency of homology in each
miRNA
family contribute to making their analysis technically challenging. This analysis
requires highly
specific assays that will allow reliable detection of individual
members and
also discriminate between mature (active) form and its precursor. In
this
presentation I will provide an overview of approaches currently
utilised in
miRNA analysis and will focus on two of them: 1) the
locked-nucleic-acids based
oligonucleotide microarray platform “miChip”, which has been developed
in
partnership with Matthias Hentze’s group and Exiqon, and qPCR for
validation of
microarray results and additional profiling of miRNAs detected by the
miChip. I will also
discuss several points
concerning preparation of samples for miRNA profiling in order to
obtain robust
profiles of miRNA expression. Validation
of Hits from an siRNA Library Screen Using
Real-Time qPCR. Shipley G.L. The Email:
gregory.l.shipley@uth.tmc.edu (micro)RNAome
of human germ cell tumors: pathological
and clinical implications. Looijenga L. Email:
l.looijenga@erasmusmc.nl Whole
miRNA Profiling from Single Embryonic Stem Cell
and early embryos. Lao K.1, Tang F.2,
Xu N.1, Livak K.1, Straus N.1, Surani
A. M.2 (1) Applied Email:
laokq@appliedbiosystems.com Teresa Rubio1,
Katrina
Academia2, Ning Liu2, Tim Wehr2, Steve
Freeby2,
Joseph Terefe1, Todd Yeck1, Aran Paulus2,
Eli
Hefner1 and Keith Hamby1, 1Bio-Rad
Laboratories, Gene Expression Division, Hercules, CA and 2Bio-Rad
Laboratories, Germany Email:
eli_hefner@bio-rad.com RNA
interference (RNAi) is a
powerful tool used to modulate gene expression and to determine gene
function.
The activation of an RNAi pathway via delivery of small interfering
RNAs
(siRNA) into cells can result in the sequence-specific degradation of a
messenger RNA (mRNA) and reduction of its corresponding protein
product. The
design of effective siRNA sequences in conjunction with efficient
cellular
delivery makes this a preferred tool for studying silencing and its
effects.
Analysis of gene specific silencing and subsequent changes in the
expression of
related genes and proteins is performed by assessing the levels of
corresponding
mRNAs or protein products. In this study, we demonstrate the effective
downregulation of the cytoskeleton protein, β-actin, using specific
27-mer
siRNAs and siLentFect Lipid Reagent in Hela cells. Subsequently, making
use of
2-dimensional gel electrophoresis (2-DGE), Capillary LC-Nanospray and
MS-MS,
Western blotting and RT-qPCR we examine changes of expression profiles
in
response to β-actin gene silencing. We will present results of our
analysis,
showing changes in the expression level of several proteins, either
directly or
indirectly associated with actin filament function. Specifically,
cofilin, an
actin filament-disassembling factor, is found to be highly
phosphorilated after
b-actin downregulation. The complimentary use of these techniques
facilitates
rapid validation of 27-mer siRNA delivery and efficacy, screening for
global
changes in protein expression and multiplex validation of targets using
qPCR
techniques. Davoren P, Miller N,
Lowery A, Mc Neill R, Kerin M. National
Breast Cancer Research
Institute, Department of Surgery, Clinical Science Institute,
University College
Hospital, Galway, Ireland Email:
pamela.davoren@gmail.com This study
aimed to validate the use
of an endogenous control gene for the normalisation of microRNA q-PCR
data in
human breast cancer tissues. The expression of five microRNAs (let-7a,
miR-10b,
miR-16, miR-21 and miR-26b) and two small nuclear/nucleolar RNAs (RNU48
and
Z30) was examined across 26 breast tumour samples, 5 benign breast
tissues and
5 normal breast tissues. A 2-step q-PCR reaction was used, the reverse
transcription
step utilising a stem-loop primer specific to each candidate RNA and
the PCR
step employing TaqMan probes (Applied Biosystems). All q-PCR was
performed on
the ABI Prism 7000 Sequence Detection System. Intra- and inter- assay
variations were all ≤ 0.3 standard deviations of a cycle threshold
value. The
two most stably-expressed candidate endogenous control genes, as
determined by
GeNorm (1) and Normfinder (2), were used to normalise the qPCR data for
the
target gene miR-30a-3p. GeNorm and
NormFinder both
identified the same single endogenous control gene as being the most
stably-expressed. The analysis methods differed in their secondary
choice of
genes, perhaps indicative of the different models used in each system.
The
relative quantity of miR-30a-3p did not differ significantly when
normalized
using the most stable gene alone or using NormFinder’s best combination
of
genes (p>0.05). Using the most stable gene alone and using GeNorm’s
recommended combination of genes for normalisation did significantly
change the
relative quantity of miR-30a-3p (p=0.001) so consideration should be
given to
using this combination of genes for a more robust normalisation. Characterization
of miRNA expression in hESC lines
using NCodeTM SYBR GreenER miRNA qRT-PCR. Uma
Lakshmipathy1, Mark
Landers1, Sam An1, 1Invitrogen
Corporation, Carlsbad,
CA, 2Burnham Institute for Medical Research, La Jolla, CA
and 3Rutgers
University, Piscataway, NJ Email:
mark.landers@invitrogen.com MicroRNAs
(miRNAs) are 19-25 nt
non-coding RNAs that regulate gene expression by inhibiting translation
or
triggering degradation of specific mRNA targets. miRNAs appear to play
a
critical role in directing cellular differentiation. Unique microRNA
expression
profiles can be associated with specific cell types and stages of
cellular
development. Further, miRNA expression profiles can vary between
undifferentiated cell lines. The NCode™ miRNA Analysis Platform
provides an
integrated solution for miRNA profiling including tools for miRNA
purification,
amplification, qRTPCR quantitation, a multispecies microarray and
labeling kit.
Using the NCodeTM SYBR GreenER miRNA qRTPCR system , we validated
global miRNA
expression profiles from several hESC lines in comparison to each
other, hEC
lines and their embryoid bodies. We were able to quantitate changes in
miRNA
expression from various cell lines by determining the fold difference
from hEC
values for critical differentiation markers. Additionally, we profiled
differentiated cardiac cell lines vs. their undifferentiated progenitor
cells
by qRTPCR to validate cardiac specific miRNA expression patterns. High-throughput
RNAi Phenotype Analysis for Cancer
Drug Target Identification and Validation by qPCR. Sukru Tuzmen, Cumhur
Ekmekci, Pinar Tuzmen, Felisa Blackmer, Holly Yin, Quick Que, Jeff
Kiefer,
David Azorsa, and Spyro Mousses Translational
Genomics Research
Institute (TGen), Email:
stuzmen@tgen.org Quantitative
real-time PCR (qPCR) is
now widely used owing to its simplicity, wide dynamic range of
quantification,
sensitivity, and precision, for accurate evaluation of gene expression.
We
describe here a qPCR process to validate specific gene silencing
triggered by
short interfering RNAs (siRNAs). RNA interference (RNAi) has become a
widely
used tool for determining the correlation between loss-of-function
phenotypes
and individual genes. Commercially available RNAi libraries have made
high-throughput genome-scale screening a feasible methodology for
studying
mammalian cell systems. However, it is crucial that any observed
phenotypic
change be confirmed at either the mRNA and/or protein level to
determine the
validity of the targeted genes. Mimicking the natural gene silencing
mechanisms
of RNA interference (RNAi), synthetic siRNAs can be used to induce
sequence-specific degradation of transcripts homologous to siRNAs. We
have used
synthetic siRNAs (Qiagen Inc., A
new method for separation and characterization of
Small RNA by On-Chip Electrophoresis. Martin Greiner 1,
Marcus Gassmann 1, Marc Valer 2, Hans Brunnert1 1Agilent
Technologies, Email:
martin_greiner@agilent.com A multitude
of microRNAs has 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
and
understanding on how RNA integrity and different purification protocols
affect
its qualitative and quantitative analysis. Here we
describe a novel Microfluidic
assay that is able to perform very sensitive high resolution analyses
of small
RNA samples on a commercial lab-on-a-chip platform commonly used for Main
Session: Single
Cell qPCR
Chair: M. Kubista / B. Rocha Lecture
hall HS 14 Raj A and Tyagi
S. Public Health
Research Institute, Email:
sanjay@phri.org Biologists
usually grind up a
tissue, extract its RNA and obtain its gene expression profile. These
profiles
represent the average number of mRNA molecules present in each cell. We
have
found that the numbers of mRNA molecules expressed by different cells
of an
identical genotype are so different from each other that very few cells
correspond to the reported averages. Using an in situ hybridization
procedure
that has a single molecule resolution we were able to explicitly count
the
number of molecules of specific mRNAs produced in each cell in a
population of
cells. We found evidence for large-scale cell-to-cell variations. Akin
to the
gene expression "noise" previously reported in the prokaryotes and
yeast, these variations stem from the nature of transcription in higher
eukaryotes, which our results indicate, occurs in bursts. Randomness in
the
onset and dissipation of these bursts of mRNA synthesis, in combination
with
the short life-time of mRNA, results in these variations. The bursts of
mRNA
synthesis in different genes occur independently of each other. The
origins of
this stochastic mRNA synthesis may lie with the unique mechanisms that
open up
chromatin context of the gene and render it conducive for mRNA
synthesis and
later sequester the gene to turn off the synthesis. If the
magnitude of the observed
variations is so large then how are cells are able to maintain their
relatively
constant phenotypes? Part of the answer is that proteins generally stay
around
in the cell longer then the mRNAs do. The preexisting pools of proteins
receive
periodic inputs from the transient bursts of mRNA production. Since the
size of
the protein pools is relatively large it is buffered against the
variations in
mRNA levels. Thus the levels of proteins vary less then the levels of
mRNAs
between cells. However, the life-times of proteins vary a lot and the
levels of
short-lived proteins must be determined by the variations in the levels
of
their respective mRNAs. To cope with such variations organisms may have
developed other yet unidentified mechanisms. In some situations these
variations
will even be beneficial, as they will serve as an extragenetic
substrate for
adaptation to the transient variations in the environment. Ruoying Tan 1,
Leila Bahreinifar 1, Dana Ridzon 1, Karl
Guegler 1,
William Strauss 2, Caifu Chen 1 1Applied
Biosystems, 850 Lincoln
Centre Dr., Foster City, CA 94404, USA and 2Department of
Molecular,
Cellular, & Developmental Biology, University of Colorado, Boulder,
CO
80309, USA Email:
Ruoying.Tan@appliedbiosystems.com We describe a
new method for
simultaneously quantifying 237 mouse microRNA (miRNA) and 21 messenger
RNA
(mRNA) genes from each of 70 single cells. The method is based on
multiplex RT,
multiplex preamplification, and singleplex real-time TaqMan® PCR
assays. Assays
are quantitative for > 3-log dynamic range. Single cell expression
signature
could classify individual ES, embryoid body (EB), and somatic cells.
Significant inter-cell variations of both miRNA and mRNA expression
were
observed within or between ES cell lines, indicating the heterogeneity
of ES
cells. Highest variability was observed among EB cells (CV = 139%),
demonstrating that EB cells undergo differentiation at different
stages. Interestingly,
expression of ES marker gene OCT4 and signaling gene Tdgf1 was absent
in 3T3
and splenocyte cells, highly expressed in ES cells, and significantly
reduced
in EB cells. Furthermore, there is no correlation in expression levels
between
miRNAs and their predicted target mRNAs, supporting translational
repression
model. Our results gain new insight of both miRNA and mRNA expression
patterns
at a single cell level. Duplex
RT-LATE-PCR reveals transcript gradients in
sets of single cells recovered from 8-cell mouse embryos. Cristina
Hartshorn, Odelya
Hartung and Biology
Dept., Email:
hartcris@brandeis.edu The formation
of two distinctive
cell lineages in preimplantation mouse embryos is characterized by
differential
gene expression. The cells of the inner cell mass (ICM) are pluripotent
and
express transcripts such as Oct4 RNA, which are down-regulated in the
surrounding, differentiated trophectoderm (TE). Conversely, other genes
are
active in the TE and silenced in the ICM. These include Xist (expressed
only in
females) and Cdx2 (in both sexes). Prior to blastocyst formation, all
these
RNAs are ubiquitously found in blastomeres of embryos at the 8-cell
stage. It
is plausible, however, that transcript levels differ among blastomeres
of the
same embryo, and that these quantitative differences may presage the
fate of
their daughter cells. Testing this hypothesis presents numerous
technical
challenges because it requires simultaneous quantification of different
RNAs in
sets of single cells isolated from the same embryo. We have overcome
these
difficulties by combining PurAmp, a single-tube method for RNA
preparation and
quantification, with LATE-PCR, an advanced form of asymmetric PCR. We
initially
constructed a duplex RT-LATE-PCR assay for real-time measurement of
Oct4 and
Xist templates and confirmed its specificity and quantitative accuracy
using
both biological samples and analysis of the LATE-PCR fluorescent
signals. The
linear slope of these signals is a sensitive tool to establish that
amplification has been achieved with comparable efficiency for all
templates
analyzed. The Oct4/Xist duplex was an ideal test system, because
comparison of
data from males and females allowed us to determine that, due to the
properties
of LATE-PCR, Oct4 amplification was unaffected by sex-related
differences in
Xist expression (females: Oct4 +, Xist ++; males: Oct4 +, Xist -). Our results
show that both Oct4 and
Xist RNA levels vary in individual blastomeres comprising the same
embryo, with
some cells having particularly elevated levels of either transcript.
This is
significant because all cells in the 8-cell embryo are believed to be
developmentally equivalent. Our data also indicate that Xist and Oct4
expression levels are not correlated at this stage, although
transcription of
both genes is up-regulated at this time in development. We have now
developed
an additional assay for simultaneous measurements of Oct4 and Cdx2 RNA,
in
light of recent findings that these two genes are reciprocally
regulated. This work
describes the first
example of RT-LATE-PCR and its utility for single-tube, multiplex
quantitative
analysis of transcripts in single cells. Levels of different RNAs can
be
accurately measured independently of their relative abundance; this is
not
possible with symmetric PCR. The techniques illustrated here are widely
applicable, for instance to gene expression analysis in stem cells and
cancer
cells and to preimplantation genetic diagnostics. We are also employing
these
strategies for multiplex quantitative end-point detection of RNA
viruses. Gene
expression or SNP profiling from picograms of RNA
using Multiplexed Tandem PCR. Corbett Life
Science, Email:
keith.stanley@corbettresearch.com MT-PCR is a
2-step PCR process for
gene profiling or mutation detection. It can be configured to produce a
gene
expression profile of up to 96 genes from 50 ng of RNA down to 10 pg of
RNA
enabling gene profiles to be obtained from a single section of
formaldehyde
fixed paraffin embedded specimens (1 to 10 ng) or from single cell
amounts of
RNA (about 10 pg). Separating the PCR reaction into 2 steps allows
optimum
conditions to be used for amplification of rare templates during the
multiplexed preamplification cycles and conditions that minimise primer
dimer
formation during the quantification stage. Expression is
first normalised
against one or more housekeeping genes included on the MT-PCR-disc, and
then
between genes on the experimental and control discs. The correlation of
MT-PCR
gene expression measurements to qPCR results, or of MT-PCR results with
100
fold different amounts of input RNA were both > 0.9. The coefficient
of
variation between assays performed on different days was 3% (in the Ct
value) for
cell line RNA (10 experiments) and 8% for FFPE sections (5 different
sections). When coupled
with High Resolution
Melt analysis, MT-PCR can be used to perform multiplexed SNP analysis
from
single cell quantities of RNA. Quantitative
RT-qPCR of individual dopaminergic
neurons from vital and fixed tissues. Birgit Liss Physiology, Email:
liss@staff.uni-marburg.de Dopaminergic
(DA) midbrain neurons
are arranged within two overlapping nuclei, the ventral tegmental area
(VTA)
and the more lateral substantia nigra (SN). Their function is crucial
for the
control of voluntary movement, reward-based behavioral decision making
as well
as for cognition and memory. Consequently, selective degeneration or
functional
dysregulation of DA neurons are causally involved in common human
disorders
like Parkinson disease (PD), drug addiction, and schizophrenia. We aim
to
identify differentially expressed genes and related cellular mechanisms
that
define different physiological and pathophysiological functions in
specific
subpopulations of DA midbrain neurons. We currently
focus on molecular
mechanisms that determine the differential vulnerability of DA neurons,
a
hallmark of Parkinson disease and its chronic animal models. To analyse
dopaminergic function and gene-expression at the level of the
individual
neurons, we combine electrophysiological patch-clamp techniques in
living mouse
brain slices, or UV-laser-microdissection (LMD) from fixed human or
mouse
brain-cryosections with quantitative real-time RT-PCR approaches. In a
hypothesis-driven approach, we focus on the role of ion channels in the
pathophysiology of the DA midbrain system. Already under physiological
control
conditions DA neurons in SN and VTA display a variety of distinct
electrophysiological properties, which are correlated with qualitative
and
quantitative differences in mRNA expression-levels of related
ion-channel
subunits. Furthermore, we could demonstrate that differences in ion
channel
expression and activity are crucial for the differential survival of DA
midbrain neurons in chronic neurodegenerative disease. In particular,
we showed
that the selective electrical silencing of DA SN neurons via
ATP-sensitive
potassium (K-ATP) channels was correlated with higher mRNA
expression-levels of
K-ATP channel subunits and lower mRNA levels of the mitochondrial
uncoupling
protein UCP-2. To identify candidate genes for differential
vulnerability of DA
neurons in an unbiased manner, we carried out single-cell microarray
studies
that resulted in a list of about sixty candidate genes that were
differentially
expressed between individual SN and VTA DA neurons. We successfully
validated
the majority (75%, n=15 of 20) of selected candidate genes from our
single-cell
microarray studies by defining their expression levels in selective and
non-amplified cDNA pools of laser-microdissected SN and VTA DA neurons,
respectively via quantitative RT-PCR. In a complementary approach, we
are
currently studying quantitative gene expression of human SN DA neurons
from
post mortem brains of idiopathic PD-patients and respective matched
controls by
combining LMD and real-time PCR as described above. Data of these
studies will
be presented and methodological issues for RT-qPCR analysis in
particular of
human post mortem material will be discussed. Radek Sindelka 1, Jiri Jonak 1,
Rebecca Hands 2, Stephen A Bustin 2, Mikael
Kubista 1,3 1IMG AS CR,
Czech Repbulic, 2Institute
of Cell and Molecular Science, Email:
sindelka@img.cas.cz Cell
determination during early
development directly depends on mRNA and protein cell content and
distribution.
In mammalian cells, mRNA profiling is limited by small amounts of RNA.
In
contract, a Xenopus egg contains very large amount of mRNA,
which opens
for expression studies on the sub cell level. Here, we quantified mRNA
levels
of several selected maternal genes in different sections of the Xenopus
egg.
We determined the amount of these mRNAs in egg sections along the
animal-vegetal axis by real-time RT-PCR. The experiments were performed
on eggs
before and after fertilization. Based on these results a 3D map of key
mRNAs in
a single Xenopus egg cell will be constructed, that will give
clues to
the relation between mRNA distribution and cell division and ultimately
differentiation. Systematic
Analysis of single cells by PCR. Mann W. Advalytix AG,
Email:
mann@advalytix.de Advalytix has
developed a new single
cell amplification platform. The AmpliGrid is a microscope slide with a
chemically modified surface in order to define 48 reaction centers
based on hydrophilic
/ hydrophobic structuring. On each of the reaction sites a 1µl
amplification
reaction can be set up. In contrast to conventional tube assays single
cells
can be deposited and quality checked immediately before the
amplification
reaction (that might be PCR, RT-PCR, etc). Combining HT methods like
FACS
sorting result in a systematic genetic analysis of single cells for
various
applications. Based on the systematic analysis of single cells
Advalytix has
developed a technique called ABC (amplification based counting) that
makes use
of the fact that in multiplex PCR there is a correlation between the
number of
drop outs and the number of identical target sequences that have been
introduced as starting material. In single cells it is now possible to
distinguish
discrete numbers of DNA or RNA sequences in an absolute manner. Detection
and quantification of mRNAs in single human
embryonic stem cells. Ståhlberg
A. Bengtsson M.
Semb H. Email:
anders.stalberg@med.lu.se Human
embryonic stem cells (hESCs)
are pluripotent cells derived from the inner cell mass of the
blastocyst. They
are unique self-renewing cells that have the capacity to generate any
cell type
in the body. This capability provides the basis for considering the
hESCs as an
unlimited source of cells for replacement therapies and for the
treatment of a
wide range of diseases such as diabetes mellitus, Parkinson and
Alzheimer
diseases. Our aim is to develop new research tools to enable a better
validation and understanding of the sequential differentiation of
pluripotent
hESC into definitive endoderm, pancreatic β-cell precursors, and
finally
-cells. Undifferentiated hESCs requirebinto
terminally differentiated expression of
NANOG, POU5F1 (also known as OCT4) and SOX2
to maintain
their unique characteristics. To date most transcriptome analyses on
hESCs have
relied on measurements from cell populations, thereby not revealing how
the
expression of NANOG, POU5F1 and SOX2 influence differentiation at a
level of
single cell. To further our understanding of the means by which these
transcription factors control the pluripotency and self-renewal of
hESCs, we
have performed quantitative gene expression studies of individual
cells.
Accurate single cell gene expression measurements require sensitive and
robust
assays. We have evaluated and optimized all steps from cell collection
to data
analysis for accurate single cell gene expression measurements. We
apply this
method for investigation of population heterogeneity, single cell
correlations
and transcript distributions. Molecular
portraiting of normal and tumor human breast
stem cells. Pece S.1, Confalonieri S.2,
Vecchi
M.2, Matera G.1, Ronzoni S.1, Tizzoni
L.2,
Bernard L.2, Pelicci P.G.1, and Di Fiore P.P.2 1IEO (Istituto
Europeo di Oncologia),
Milan, Italy and 2FIRC Institute for Molecular Oncology
(IFOM),
Milan, Italy Email:
salvatore.pece@ifom-ieo-campus.it A prediction
of the stem cell theory
of breast cancer is that complete elucidation of the normal stem cell
biology
will be instrumental to gain insights into breast carcinogenesis. Given
the
lack of specific human breast stem cell markers, we devised a strategy
based on
the ability of breast stem cells to generate clonally-derived
‘mammospheres’ ex
vivo, in combination with the use of a ‘surrogate’ marker, the PKH26
cell
linker, which stably incorporates a fluorescent dye into the lipid
regions of
the plasma membrane. The strength of this approach is that stem cells
are not
defined phenotypically, i.e. using cell surface markers, but
functionally
through their intrinsic property to be slow-dividing. In fact, they
accumulate
the PKH26 dye and remain the most intensively fluorescent cells within
mammospheres, whereas their actively dividing and differentiating
progeny
progressively loose fluorescence through dilution of the membrane-bound
dye. We
used FACS analysis to sort the most highly fluorescent cells,
comprising the
strict-sense stem cells, from the progeny of committed progenitors.
Functional
analysis of the different fractions confirmed that only the PKH26+
cells
exhibited key defining features of ‘stemness’, such as: i) self-renewal
property (re-formation of mammospheres upon serial passages in vitro);
ii)
ability to generate both epithelial and myoepithelial histotypes, as
determined
with specific lineage markers; iii) formation of alveolar/ductal-like
outgrowths in vitro. Using oligonucleotide-based arrays (Affymetrix),
we
obtained trascriptional profiles of the two populations separated by
their
differential epifluorescence. The comparative analysis of genes
differentially
regulated by a factor ≥2 uncovered many distinguishing features between
the two
PKH26 populations, strengthening the notion that we indeed separated
stem cells
from their differentiating progeny. Selected candidate hits were
further
validated by Q-RT-PCR using a multiplex reaction with pooled 96 Taqman
gene
expression assays (Applied Byosystem) as a source of primers in a
pre-amplification reaction, followed by a Taqman low density array
experiment.
This approach confirmed that genes associated with an immature and
quiescent
state (‘stemness’) were associated with PKH26+ cells. In contrast,
PKH26- cells
over-expressed transcripts associated with proliferation, cell cycle
progression and checkpoint control together with markers of
myoepithelial/epithelial differentiation. In conclusion, we set up a
strategy
for the functional characterization and molecular portraiting of normal
(and
tumor) breast stem cells, with the ultimate goal to highlight the
molecular
mechanisms controlling normal and aberrant morphogenetic programs. Quantification
of multiple gene expression in individual
cells. Antonio Peixoto,
Marta Monteiro, Benedita Rocha,
Henrique Veiga-Fernandes INSERM U591,
Faculty of Medicine Email:
rocha@necker.fr Quantitative
gene expression
analysis aims to define the gene expression patterns determining cell
behavior.
So far, these assessments can only be performed at the population
level.
Therefore, they determine the average gene expression within a
population,
overlooking possible cell-to-cell heterogeneity that could lead to
different
cell behaviors/cell fates. Understanding individual cell behavior
requires
multiple gene expression analyses of single cells, and may be
fundamental for
the understanding of all types of biological events and/or
differentiation
processes. We here describe a new reverse transcription-polymerase
chain
reaction (RT-PCR) approach allowing the simultaneous quantification of
the expression
of 20 genes in the same single cell. This method has broad application,
in
different species and any type of gene combination. RT efficiency is
evaluated.
Uniform and maximized amplification conditions for all genes are
provided.
Abundance relationships are maintained, allowing the precise
quantification of
the absolute number of mRNA molecules per cell, ranging from 2 to 1.28
x 10(9)
for each individual gene. We evaluated the impact of this approach on
functional genetic read-outs by studying an apparently homogeneous
population
(monoclonal T cells recovered 4 d after antigen stimulation), using
either this
method or conventional real-time RT-PCR. Single-cell studies revealed
considerable cell-to-cell variation: All T cells did not express all
individual
genes. Gene coexpression patterns were very heterogeneous. mRNA copy
numbers
varied between different transcripts and in different cells. As a
consequence,
this single-cell assay introduces new and fundamental information
regarding
functional genomic read-outs. By comparison, we also show that
conventional
quantitative assays determining population averages supply insufficient
information, and may even be highly misleading. Quantitative
PCR of heterogeneous tissue: Lessons from
the islets of Langerhans. Martin Bengtsson1, Anders
Ståhlberg1,
Patrik Rorsman2 Email:
martin.bengtsson@med.lu.se The islets of
Langerhans reside in
the pancreas where they serve as the glucose sensor of the body, making
sure
the blood glucose concentration stays within a healthy range. This
regulation
fails in patients with diabetes. Each islet consists of approximately
1000
cells, of which ~80% are insulinproducting b-cells, ~20%
glucagonsecreting
a-cells and ~5% somatostatinreleasing d-cells. The functions of these
cells are
fundamentally different, and studies of them require either cell
sorting or
single cell analysis. In our search to reveal cellular mechanisms and
find
possible therapeutic targets for diabetes treatment we developed a
method for
quantitative mRNA measurements in individual mammalian cells using
RT-PCR. We
took great care to maximize the robustness, reliability and efficiency
of the
method. For example, we evaluated the need for – and choice of – cell
lysis
buffer and heat treatment. We investigated the accuracy of the
quantification,
thereby defining a limit at which the technical variation exceeds the
biological variation. These results, together with data from glucose
stimulation of islet cells, will be showed in the presentation. Islet cells
are electrically active.
Ion channels play a fundamental role in hormone release and several
types of
diabetes are caused by dysfunctional ion channels. In collaboration
with
Applied Biosystems, we combined electrophysiological recordings, single
cell
collection and the TaqMan® PreAmp Master Mix Kit on individual
islet cells. The
preamplification allows us to measure hundreds of genes from a single
cell, and
we investigated the isoforms of sodium ion channels in a- and b-cells.
The
results revealed cell-type specific expression of sodium channel
isoforms and
correlations to Na2+-currents measured with patch-clamp. Quantitative
real time PCR for single tumor cell based
diagnostics. Kemming, D.
Meyer-Staeckling, S. Alpers, UKE Email:
d.kemming@uke.uni-hamburg.de One third of
annually 50,000 newly
diagnosed breast cancer patients will die from metastases. However, the
individual risk for metastases is still estimated statistically by
parameters
determined on the primary tumor yet. We and other research groups have
shown
that breast cancer patients harbor single disseminated tumor cells
(DTC) in
their bone marrow or peripheral blood even in the absence of lymph-node
involve-ment (stage N0) or distant metastases (stage M0) at the time of
primary
surgery. The detection of aberrations of key oncogenic genes could
enhance the
specificity and may lead to the identification of the potential founder
cells
of metastases. In
consequence, we developed a
method for the isolation and real time PCR based characterization of
tumor
cells. The cells to be analyzed are transferred under micro-scopic
control
selectively on hydrophobic coated glass slides carrying lysis buffer in
a spot.
The surface ensures that the genetic material can be completely removed
after
the cell lysis. This lysate is subsequently subjected to isothermal
whole
genome am-plification (WGA). As a test system MDA-MB-468 and SK-BR-3
cells were
used, ge-nomic leukocyte DNA was used as reference material. MDA cells
show a
strong am-plification of the EGF receptor gene while SK-BR-3 cells show
a low
level amplifica-tion of this gene. Single cell WGA products were used
to
analyze EGFR gene ampli-fication by real time PCR. The copy number of
the SOD2
gene was used as refer-ence. In order to test if WGA products reflect
the
genomic situation of the tumor cells, EGFR gene copy numbers were
compared
between WGA products and tumor cell DNA. Using two different cell
lines, we
were able to determine similar genomic altera-tions in the WGA products
and in
the unamplified material. The
specificity of the method was
confirmed by sequencing and microsatellite analy-ses. A cell of a cell
line
harbouring two known mutations in the tp53 gene was sub-jected to the
single
cell picking/WGA procedure. Both mutations were found using the genetic
material isolated from the single cell. PCRs targeting microsatellites
on
chro-mosome 7, 8, 10, 13, 16 and 17 were performed to ensure the
amplification
of the entire genome. Therefore
this method might be
suitable for more detailed examinations of bone marrow and blood from
cancer
patients. Heterogeneity
in complex tissues identified by
quantification of nucleic acids in single cells. Philip Day1,2, Lin Chen1,
Pierre-Alain Auroux2, Stephan Mohr2, Nicholas
Goddard2,
Andreas Manz1 and Peter Fielden2. 1Analytical
Sciences, ISAS, Email:
philip.j.day@manchester.ac.uk Real-time
quantitative and
conventional end-point polymerase chain reaction (PCR) are ubiquitously
applied
in gene-based assays, however routine reproducibility is limited by
intrinsic
technical limitations. These restrictions include poor compatibility to
study
low transcript number amongst a high background of other nucleic acids,
analysis of several targets from minute biopsy samples, single cell
analyses,
plus additional constraints relating to time, expense and risk of
cross-contamination. PCR micro
total analysis systems
(μTAS) devices can circumvent these drawbacks. Two uTAS concepts are to
be
presented. In the first, a novel approach is based on a shunting system
where
an aqueous sample plug is shunted from one temperature zone to another
by a
syringe pump system, and benefits from reaction parallelisation. A
second more
advanced system engages the application of flowing streams of aqueous
nanolitre
droplets for use in single cell PCR. Cell lysis, reagent mixing,
thermal
cycling and microfabricated real-time optics have been developed. These
will
ultimately derive μTAS devices for very high throughput uses and more
robust
inter-laboratory standardisation. Data is
presented revealing that
quantification of nucleic acids via PCR has a mandatory requirement to
employ
miniaturisation, and that this will assist in functionally relating
numbers of
copies of analyte nucleic acid to single cell type and decipher the
cell
composition of heterogeneous tissues.
Session:
Immuno - qPCR
Chair: HHD. Meyer / C. Niemeyer Lecture
hall HS 14 Niemeyer
C. Universität
Dortmund, Germany, FB Chemie, Biologisch-Chemische
Mikrostrukturtechnik,
Otto-Hahn Str. 6, D-44227 Dortmund, e-mail:
christof.niemeyer@uni-dortmund.de Email:
sekretariat-bcmt.chemie@uni-dortmund.de The
Immuno-qPCR technology combines
the advantages of flexible and robust immunoassays with an exponential
signal
amplification, typical for PCR [1]. Immuno-qPCR is based on chimeric
conjugates
of specific antibodies and nucleic acid molecules, the latter of which
are used
as markers to be amplified by PCR for signal generation. The enormous
efficiency of nucleic acid amplification typically leads to a 100 –
10,000 fold
increase in sensitivity, as compared with the analogous
enzyme-amplified immunoassay.
We have developed a proprietary detection and screening technology
focused on
the analysis of low abundant and hardly detectable biomarkers. This
technology
is being commercialized under the trademark Imperacer™ by the The lecture
provides an overview on
the scope and performance of the Imperacer™ Immuno-qPCR technology,
including
detailed discussions of experimental parameters, such as sensitivity
and
dynamic range, sample requirements, and tolerances against matrix
effects in
the detection of various biomarkers, including drug candidates,
small-molecule
hormones, cytokines, tumormarkers, viral proteins, and others. Use
of Immuno-qPCR for quantifying proteins in
large-scale TAP-tag collections. Lind K. and Norbeck
J. Email:
kristina.lind@chalmers.se In recent
years, the sequencing of
several eukaryotic genomes has paved the way for truly large-scale
experimental
approaches. Both for our understanding of the biological process in
cells and
for the development of new drugs. Saccharomyces cerevisiae, was the
first
completely sequenced eukaryot and is a widely used model organism.
Several
genome-wide collections have been constructed in S.cerevisiae, e.g. a
set of
gene-deletions covering all non-essential genes, GFP-tag (Green
Fluorescent
Protein-tag) collections and TAP-tag (Tandem Affinity Purification-tag)
collections for essentially all 6000 genes. The GFP-collection has been
used in
combination with flow cytometry (FACS) to quantify the expression of
all tagged
proteins. Similarly, the TAP-tag collection has been used in
combination with
western blotting for protein quantification. Both these approaches have
serious
drawbacks, the GFP-approach suffers from a rather high detection limit
and the
TAP-tag approach is limited by the difficulties associated with protein
extraction and western analysis (e.g. difficulties in background
subtraction
and lack of linearity because of saturation of signal). Thus, there is
a need
for development of more sensitive and reliable protein quantification
methods
suitable for large-scale analysis. We have
adapted the immuno-qPCR
assay, previously used by us for quantifying prostate specific antigen
(PSA),
for quantification/detection of the TAP-domain. Briefly, we utilize an
affinity
purified chicken antibody raised against protein A (which is present in
two
copies in the TAP-tag) as both capture and DNA-conjugated detection
antibody.
The immuno-qPCR-detection of the TAP-tag is a sensitive method with a
large
quantification range that combines the molecular specificity of
antibodies with
the DNA amplification power of PCR. Using real-time PCR there is no
need for
gel electrophoresis which makes the assay time short. A drawback
however,
compared to western blot analysis, is that no information about protein
modifications or breakdown products is gained using immuno-qPCR. We
envisage
that our assay, when combined with automated sample handling, can be
used to
sensitively quantify essentially all yeast proteins under a variety of
conditions. Furthermore, our TAP-assay is not restricted to use in
yeast, the
TAP-tag has proven its worth in organisms ranging from E.coli to humans. With this
newly developed assay we
have analysed the expression of a number of proteins in yeast grown on
YPD or
YPD plus 1 M NaCl and compared it to published data from 2D-PAGE and a
large
scale western approach. The correlation to the 2D-PAGE data was good
while the
correlation to western data (restricted to YPD-growth) was more
problematic. We
will discuss the reasons for this. Immuno-Real
Time-PCR as a sensitive diagnostic tool:
case of prion proteins. Ruelle
Virginie and ElMoualij
Benaissa 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. Prion diseases are caused by
tertiary
conformational change of the normal form of prion protein (PrPc) in
host cells
to the pathologic form (PrPsc) and its accumulation in central nervous
system2. The
conformational change of the
pathologic form confers to it a partial protease resistance property
being very
convenient to distinguish the two prion forms, normal and pathologic.
Actually,
different rapid detection kits are approved by the European Community
for a
systematic diagnosis of bovine spongiform encephalopathy and scrapie,
by
screening the brain stem at the post-mortem stage3. These
detection kits
are very efficient to detect prion protein at the post-mortem stage and
permitted to warrant the safety of the bovine and sheep production.
However,
other ultra-sensitive methods need to be developed to allow an early
detection
of prion protein for living animals. In this
study, the
immuno-quantitative-Polymerase chain reaction (iqPCR)4 was
applied
to detect ultra-low levels of prion protein. 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
format5-6.
To illustrate the advantages of iqPCR, we have compared it with a
conventional
ELISA and western blotting technique in experiments aimed at detecting
the
resistant form of prion protein in bovine and human brain extract.
Using iqPCR,
a minute quantity of prion was detected with a detection threshold at
least
10-fold lower than classical techniques. The iqPCR could therefore be
potentially useful for the detection of prion protein at early stage. Feasibility
of simultaneous measurements of mRNA
expression and corresponding protein level in microdissected tissue
samples by
real-time technology: PSA in normal and tumour tissues as a
demonstrative
model. Pamela Pinzani 1, Kristina Lind 2,
Francesca Malentacchi 1, Francesca Salvianti 1,
Mikael
Kubista 3, Mario Pazzagli 1, Claudio Orlando 1. 1Department of
Clinical
Physiopathology, Clinical Biochemistry Unit, University of Florence,
Italy, 2Department
of Chemistry and Bioscience, Chalmers University of Technology,
Gothenburg,
Sweden and 3TATAA Biocenter AB, Gothenburg, Sweden Email:
p.pinzani@dfc.unifi.it Laser
assisted microdissection (LAM)
has been introduced extensively in cancer molecular biology studies
with the
aim of selecting pure cell populations from heterogeneous tissues. New
technologies in the field of LAM can thus overcome the problem of
cellular
heterogeneity that represents a significant barrier to the molecular
analysis
of normal and pathological tissue (Simone et al 2000). Moreover, it
allows
molecular analysis of cell populations in their native tissue
environment and
it may be potentially applicable to biopsies obtained in preoperative
diagnostic procedures. Several attempts have been made in order to
determine
whether mRNA levels could be used to accurately predict protein levels
in
tissue sample extracts (Hiser et al. 2006). Results obtained for
different
proteins gave either positive or negative results (Gygi et al. 1999;
Nicoletti
et al. 2001) with an additional and intrinsic limitation due to the
relative
lack of sensitivity of the actual methods used for protein measurements. Recently,
immuno qPCR has been
developed as a new tool for the measurement of proteins levels with up
to
1000-fold increase of detection limit compared to the commonly used
immunoassay. It is based on the use of a real-time PCR detection of the
immuno-complex generated in the immunoassays. It has been applied to
the
measurement of protein in serum and plasma samples, but until now no
evidence
has been reported on microdissected tissues. In this
study, prostate-specific
antigen (PSA) mRNA expression data were generated from microdissected
primary
prostate cancers and corresponding normal tissues by real-time RT-PCR.
To
calculate the expression of PSA mRNA in each microdissected sample, we
referred
to an external reference curve generated with synthetic cDNA obtained
by
cloning the target in the expression vector pcDNA3.1/CT-EGFP-TOPO
(Invitrogen).
PSA protein determinations were performed at Chalmers University of
Technology
( In
conclusion, we demonstrated for
the first time the feasibility of the simultaneous application of
real-time
RT-PCR and immuno qPCR to purified homogeneous cell populations
obtained by
LAM. These methods could represent a powerful tool to enhance the
diagnostic
value of gene expression studies in human cancers at mRNA and protein
levels. Session: Pre-analytical-Steps
Chair: A. Stahlberg / J. Huggett Lecture
hall HS 15 An
optimised protocol for extracting RNA from single
bovine oocyte and blastomeres. Marc Boelhauve1, Fabiola F
Paula-Lopes2, Tuna Güngör1, Eckhard Wolf1 1Institute of
Molecular Animal
Breeding and Biotechnology, LMU Munich, Germany and 2Laboratório
de
Biotécnicas da Reprodução, Departamento de
Medicina Veterinária da Universidade
Federal Rural de Pernambuco, Recife – PE, Brazil Email:
m.boelhauve@gen.vetmed.uni-muenchen.de Quantitative
PCR (qPCR) analysis of
gene expression in single bovine oocytes or blastomeres from early
preimplantation embryos needs to meet optimized requirements for the
isolation
of RNA, reverse transcription reaction and even qPCR. In this study the
first
hurdle to a sufficient detection of low abundant genes was analyzed.
There are
many protocols available for extracting RNA from different materials,
but normally
large amounts of tissues are required. For extracting RNA from bovine
preimplantation embryos several methods are available, but the RNA
recovery per
embryo is too low for the detection of low abundant genes. Therefore a
modified/optimised isolation method is essential for obtaining
sufficient RNA
recoveries of single oocytes/embryos or even single blastomeres.
Several
experiments were conducted to increase the RNA recovery. In the first
experiment different isolation protocols, taken from the literature,
were
compared (isolation of total RNA and messenger RNA). In a second
experiment the
best isolation protocol was used to analyse the influence of a
coprecipitant
(e.g. glycogen) on the RNA recovery and the inhibition of the reverse
transcription process. In the third experiment the collection/storage
of
oocytes was compared (e.g. RNAlater or liquid nitrogen). Due to the low
amount
of RNAs, standards for the RNA isolation and the reverse transcription
process
were evaluated. Finally, total RNA concentrations were measured by two
different methods (Nanodrop and Agilent Bioanalyzer) of ten bovine
embryonic
developmental stages (immature oocyte up to hatched blastocyst). For
each
experiment studied, RNA was reverse transcripted with Omni Extreme
Script and random
hexamer primers. The RNA recovery was analysed by the detection of
transcript
levels of high (STAT3), middle (Histone 2A) and low abundant (Leptin
Receptor,
LEPR) genes in an ABI PRISM SDS 7000 apparatus. Laser
microdissection – Bridging the gap between
sample preparation and molecular biological analysis. Hagen-Mann
Kerstin Carl
Zeiss MicroImaging, Germany Email:
k.hagen-mann@zeiss.de Targeting
single cells, defined
cells of a united cell structure or cell culture requires tools for
precise
selection of such targets. By means of laser microdissection and
contact free
catapulting into reaction tubes or onto glass surfaces researchers can
prepare
very homogeneous samples for gene expression, fingerprinting or pure
cell
cultures. We can prove that manipulation with laser microdissection
does not
affect the behaviour of cells in a cell culture, it does not change
their
characteristics (e.g. markers) and they survive this treatment easily.
The target
for laser microdissection can be an area of a tissue section, single
cells of
such a slide or even nuclei and single chromosomes or parts of them as
well as
living cells. Because the excision and transfer into the reaction tube
is
contact free, this technique provides a perfect contamination free
environment
for preparation of samples for all contamination sensitive downstream
analysis
applications. Reproducible sample preparation of defined numbers of
cells are
the prerequisite for quantitative experiments and their results. Bring
in the marines! Removal of contaminating DNA by
marine enzymes in RT-PCR. Elde M., Lanes O.
and
Gjellesvik D.R. Email:
morten.elde@biotec.no Marine
enzymes from cold-adapted
organisms exhibit properties that make them useful as tools in
molecular
biology. A common feature for these enzymes is high activity at low
temperatures and that they are easily heat inactivated at moderate
temperatures. Here we present the use of two different marine enzymes
in RT-PCR. The presence
of contaminating DNA in
quantitative RT-PCR is often a problem and can give erroneous results.
The
origin of the contaminating DNA may be genomic from the RNA source, or
may be
previous PCR products (carry-over contamination). A nuclease
from the arctic shrimp ( Pandalus
borealis ) has properties that makes it useful for removal of
contaminating
DNA in a RT-PCR reaction. It is double-strand specific and easily
inactivated.
Here we show how the shrimp nuclease, in a one-step RT-PCR, removes
double-stranded DNA from the RT-PCR mix, leaving RNA and primers intact. Uracil-DNA-Glycosylase
(UNG) is an
enzyme often used to remove carry-over contaminants in quantitative
PCR. We
have tested the use of a marine UNG from cod ( Gadus morhua )
for carry-over
prevention in RT-PCR, and compared it with other UNGs already
available.
Contaminating DNA is efficiently removed by the cod UNG but does not
affect the
sensitivity (Ct) of the qRT-PCR assay. Multiplex
preamplification of limited samples and novel
analytical controls. Zimmermann
Bernhard,
Wang Jianghua, Wong
David UCLA, Dental
Research Institute Email:
bgz@ucla.edu We present a
new method for the
multiplex 1-step RT-PCR based preamplification of mRNA that allows the
determination of extensive expression patterns even from limited
clinical
samples such as saliva, where sample volume, abundance and integrity of
mRNA
are limited. The singleplex real-time PCR analysis following the
preamplification can be performed with cost effective dye based
chemistries in
low reaction volumes with assay performance equal to conventional
RT-qPCR. We
show that the analysis can even be performed in 33 nanoliter reaction
volumes
on the BioTrove Open Array platform, which will enable high-throughput
quantification in previously unparalleled extent, carrying out over
3000
individual qPCR reactions on a single slide. In addition
to the analytical
targets, multiple transcripts for normalization can be integrated in
the
analysis. As these are reverse transcribed and amplified in the same
reaction
with the analytical targets, this also reduces the variability of the
measurements to a large extent. Furthermore we present the use of
several
exogenous mRNAs as controls for sample analysis. These can be spiked
into the
sample prior to extraction or during the analytical process, thus
controlling
for inhibition, extraction efficiency and analytical variability. In
the future
they will also be used as semi-quantitative internal standards. Finally our
work indicates the
necessity to incorporate appropriate carrier material such as tRNA into
the
handling of samples and reference material of low RNA concentration. Overall, the
new method allows the
flexibility to establish extensive expression patterns of well over 30
targets
for all kinds of biological samples and RNA concentrations. The high
specificity and the implementation of extensive controls present a
major
advancement for research and clinical analysis of nucleic acids by qPCR
and
also other downstream methods. Sybille
Matthey1, Vlad
Popovici2, Janine Antonov1, Andrea Oberli1,
Anna Baltzer1, Mauro Delorenzi2 and Hans
Jörg Altermatt3
and Rolf Jaggi1 1Department of
Clinical Research,
University of Bern, CH-3010 Bern, Switzerland, 2Swiss
Institute of
Bioinformatics (SIB), CH-1015 Lausanne, Switzerland and 3Pathology
Länggasse, CH-3012 Bern, Switzerland Email:
rolf.jaggi@dkf.unibe.ch Expression
profiling with DNA chips
is very popular and widely used in several areas of research including
breast
cancer. Until now, this technology depended on intact or at least good
quality
RNA which can only be isolated from fresh or freshly preserved tumor
material
(e.g. by snap freezing tumor material). Such material is not collected
on a
regular basis and therefore, only exists in very limited sets of
samples. On
the other hand, many thousands of samples exist as Formalin-fixed,
Paraffin-embedded (FFPE) blocks, as they are collected and preserved in
routine
diagnostics. Even more interesting are samples, which were collected in
the
context of clinically controlled studies. Unfortunately, formalin
fixation and
paraffin embedding does not only lead to partial degradation of RNA, it
also
leads to extensive chemical cross-linking between nucleic acids (intra-
and
intermolecular) and between nucleic acids and proteins. As a result,
RNA can
only be isolated from tissue homogenates after extensive digestion with
proteinase. We developed a special de-modification procedure which
reverts
N-methylol bonds formed during fixation between nucleotide bases and
formaldehyde and which hydrolyzes methylene bridges between nucleic
acids. The
resulting RNA is highly suited for down-stream applications, e.g.
TaqMan
expression assays when used in combination with gene-specific cDNA and
optimized TaqMan assays. The same RNA was also used for DNA chip
analyses: RNA
was amplified in a single round of random primed, T7-tagged reverse
transcription followed by in vitro transcription in the presence of
biotinylated or Cy3-labeled nucleotides. The resulting cRNA was
hybridized to
12k CombiMatrix arrays and 44k Agilent arrays. CombiMatrix uses
35-40-mer
oligonucleotides synthesized in a special porous layer, Agilent uses
60-70-mer
oligonucleotides synthesized on glass slides. The technical quality of
chips
was assessed by comparing the same probe on different arrays (technical
replicates). More interestingly, expression values from FFPE-derived
RNA were
compared to expression values from intact RNA (derived of snap frozen
material
of the same tumors). Correlations between such matched samples were in
the
range of 0.80-0.95, depending on the gene list of interest. We also
compared
DNA chip with QPCR for a number of genes: again, good correlations were
observed between QPCR and DNA chips for both, good quality RNA (snap
frozen)
and FFPE-derived RNA. We have
developed a rapid and simple
procedure for RNA isolation from FFPE material. The resulting RNA is
suitable
for QPCR and DNA chip based analyses. Samples from a clinical study on
the
efficacy of Tamoxifen and Letrozole for which only FFPE blocks exist,
are
currently being analyzed. Simple
& effective measures to increase
consistency from sample to Ct. Kavanagh I. Thermo Fisher
Scientific, Email:
ian.kavanagh@thermofisher.com There are
many choices a user must
make in order to establish a viable QRT-PCR procedure. At Thermo
Scientific
(ABgene) we have identified aspects within a QRT-PCR protocol, ranging
from
sample preparation through to amplification, which can introduce
variability to
data. Introduction of simple, but effective measures can result in
increased
consistency throughout QRT-PCR. During sample
preparation contamination
of an RNA sample with genomic DNA can lead to decreased reaction
efficiency and
false positives. DNase I is commonly used for removing DNA
contamination, but
this method is both time-consuming and increases the risk of RNA
degradation.
This degradation can occur during the harsh inactivation conditions of
the
DNase I. We have developed an alternative method that degrades
contaminating
DNA, which is equal in performance to DNase I, but also saves time and
effort. A choice of
priming strategy must be
made to initiate reverse transcription (RT). We have assessed the
effect that a
variety of alternative RT priming methods can have on the sensitivity
of QPCR
in different applications. We found that using a gene-specific primer
for RT
demonstrated the most sensitive QPCR amplification, with earlier Ct
values.
However, gene-specific priming limits the number of cDNA pool
applications. We
found that a blend of anchored oligo-dT and random hexamers generated
the best
Ct and end-point readings. In addition, this blend allows greater
flexibility
in the choice of endogenous control genes. During the
amplification step of
QRT-PCR, dUTP is normally used in conjunction with the enzyme
Uracil-N-Glycosylase (UNG) in order to remove any amplicon carry-over
contamination. When using dUTP, higher Ct values and decreased QPCR
reaction
efficiencies of up to 5% are generated. Therefore a further way to
increase
consistency is to use a master mix that contains dTTP instead of dUTP. Thermo
Scientific has discovered
that the use of white polypropylene plastic consumables can eliminate
inefficient signal reflection observed in natural (clear) or frosted
polypropylene. Different shades of white were tested to determine the
optimum
colour to provide the most sensitive detection. Despite the benefit of
generating more reliable data, one of the biggest problems when using
white
plates has been visualizing the master mix in the well. One of our
recently
launched products, ABsoluteTM Blue QPCR master mix, solves
this
problem. This QPCR master mix contains an inert blue dye to
significantly
enhance the contrast between reagent and plastic and make verification
of
master mix dispensing quick, easy and foolproof. The most
important aspect of QPCR is
increasing the consistency and reproducibility of the results
generated. By
employing a number of simple yet effective measures, users can
significantly
reduce many of the variables inherent in the QPCR process. Pre-amplification
with standardized mixtures of
internal standards enables highly multiplexed Quantitative PCR analysis
when
sample size is limited or restricted by the volume requirements of
nanofluidic
systems. James C.
Willey1, Erin, L.
Crawford1, Charles Knight2, Bradley Austermiller2 1University of
Email:
james.willey2@utoledo.edu There is
increasing need to quantify
transcript abundance of many genes in small samples such as single cell
samples
in stem cell differentiation experiments, or needle biopsy or
microdissected
samples in diagnostics. In addition, in nanofluidic systems input
sample volume
may be limited to the point where signal is lost. Signal loss occurs
because no
transcript molecules are present, not because the method is limited
with
respect to signal amplification. One way to address this is sample
pre-amplification. Pre-amplification may increase the number of
transcript
molecules representing a particular gene by many orders of magnitude.
However,
if the inter-transcript variation in PCR amplification is not
controlled, two
rounds of amplification may increase analytical variation in relative
representation of the transcripts representing different genes. In this
study,
Standardized RT (StaRT)-PCR was used in two rounds of amplification of
96 genes
in the Stratagene Universal Reference RNA. In the first round, cDNA was
mixed
with a standardized mixture of internal standards (SMIS) containing an
internal
standard for each of the 96 genes and primers for each of the genes and
PCR
amplified for 35 cycles. Next, products from round one were diluted,
aliquoted,
and each aliquot was combined with primers for one gene, and amplified
for an
additional 35 cycles. No additional cDNA or SMIS was added. Expression
values
obtained from this method were highly correlated with values obtained
by
typical one round amplification (R = 0.993, p < 0.001). Products
from round
one could be diluted as much as 100,000-fold and still be quantified
following
round two amplification. Thus, using this method 96 genes were measured
in the
same amount of cDNA typically used to measure one gene with one round
of PCR.
This method was then successfully implemented in the high throughput
Standardized Expression Measurement (SEM) Center and applied to
quantify
transcript abundance in a series of transthoracic fine needle aspirate
biopsies
from suspected lung cancers. This method should enable wider
application of
nanofluidic systems to quantify transcript abundance by quantitative
RT-PCR and
increase the number of clinical samples that may be assessed. J.C.
Willey has
equity interest in and is a consultant for Gene Express, Inc. E.L.
Crawford has
equity interest in Gene Express, Inc. Session:
qPCR
BioStatistics & BioInformatics
Chair: M. Pfaffl / T. Bar Lecture
hall HS 15 Michael W.
Pfaffl Lehrstuhl
fuer Physiologie;
Physiology - TU München, Email:
michael.pfaffl@wzw.tum.de Quantitative
real-time RT-PCR
(qRT-PCR) is widely and increasingly used in any kind of mRNA
quantification,
because of its high specificity, extreme sensitivity, reliable
reproducibility,
and wide dynamic quantification range. While qRT-PCR has a tremendous
potential
for analytical and quantitative applications, a comprehensive
understanding of
its underlying principles is important. Beside the classical RT-PCR
parameters,
e.g. primer design, RNA quality, RT and polymerase performances, the
fidelity
of the quantification process is highly dependent on a valid data
analysis. This review
talk covers all aspects
of data analysis in real-time PCR: data acquisition and threshold cycle
determination procedures, e.g. trueness, reproducibility, and
robustness of
fluorescence readout; the potentials of data modification by the cycler
software, e.g. curve smoothing, background correction, data adjustment/
normalization, and generation of standard curves. The talk will focus
particularly on relative quantification methods using efficiency
corrected
quantification models. Furthermore useful and new bioinformatical,
biostatical
as well as multi-dimensional expression software tools will be
presented. Detection
of defective PCR samples with module Outlier
of Kineret software. Bar T., Tichopad A.
and Dahan E. Email:
tzachi.bar@labonnet.com For proper
quantification with
real-time PCR compared samples should have similar kinetics. However,
inhibition of PCR is common when working with biological samples and
may invalidate
this assumption. Herein we present the Outlier function of Kineret,
software
for real-time PCR data analysis, management and validation. Outlier
consists of
two parts, kinetics outlier detection and CT-outlier detection. The
kinetics
outlier detection is based on multivariate analysis of geometric
properties of
amplification curves. Testing this function, we could identify 80-100%
of
artificially inhibited samples with significantly aberrant kinetics.
The
alteration of the kinetics was associated with a shift of about half
cycle. The
CT-outlier detection function utilizes the predefined experimental
design,
including definition of replicates, to detect PCR with outlying CTs.
Intersecting the findings of the two methods results in robust
validation tool
for real-time PCR that enables evidence-based decision making in
research and
diagnostics. Hellemans Jan,
Vandesompele
Jo Center for
Medical Genetics, Email:
Jan.Hellemans@UGent.be Since its
introduction more than 10
years ago, qPCR has become the standard method for quantification of
nucleic
acid sequences. To address the shortcomings of the available
quantification
strategies (and software tools), we developed a quantification
framework based
on multiple reference gene normalization, inter-run calibration and
proper
error propagation along the entire calculation track. Two different
experimental set-ups
can be followed in a qPCR relative quantification experiment: sample
maximization (recommended approach) or gene maximization (to be used in
prospective studies). Whatever set-up is used, inter-run calibration is
required to correct for possible run-to-run variation whenever not all
samples
for a given gene are analyzed in the same run. To this purpose, the
experimenter needs to analyze so-called inter-run calibrators
(identical
samples that are tested in both runs). By measuring the difference in
quantification cycle or normalized relative quantity (NRQ) between the
inter-run calibrators in both runs, it is possible to calculate a
calibration
factor to remove the run-to-run difference. Inter-run calibration based
on
NRQ's has the important advantage that independently prepared cDNA of
the same
RNA source (or a biological replicate) can be used as a calibrator in
the
different runs. In contrast, calibration based on Ct values should be
used if
the inter-run calibrators can not be normalized (e.g. plasmid DNA). We
present
advanced algorithms, using imputation of missing data, to allow
inter-run
calibration for all types of experimental design. Relative
quantification using the
delta-delta-Ct formula requires the selection of a reference sample (or
so-called within-run calibrator). In principle any sample, or even an
arbitrary
cycle value can be chosen as a reference. The choice of reference cycle
value
does not influence the relative quantification result, but profoundly
affects the
final error on the relative quantities if the error on the estimated
amplification efficiency is propagated in the calculations. We
demonstrate that
the overall final error can be minimized by selecting the average Ct
value of
all samples for a given gene as a reference for that gene. The proposed
calculation framework
improves on the existing quantification strategies to provide more
reliable
results, and a better estimate of their error. To address the problem
of the
vast number of calculations, we partially implemented this framework in
the
existing qBase software [1]. The complete framework will soon be
available in
Biogazelle's platform independent and powerful qBasePlus software,
providing an
intuitive user interface for streamlined processing of all kinds of
qPCR data. Real-time
PCR Expression Profiling. Kubista Mikael TATAA Biocenter,
Sweden Email:
mikael.kubista@tataa.com 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
proper experimental design is required as well as methods to extract
information from the measured data. In my talk I will present some of
the first
real-time PCR expression profiling studies, starting from the
collection and
pre-processing of real-time PCR data, to the classification of samples
as well
as genes with some of the most powerful methods available
(www.multid.se).
These approaches, which still are in their infancy, are expected to
become the
golden tool in future drug discovery, molecular diagnostics and
prognostics, as
well as in basic research in molecular cell biology and molecular
physiology. 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 Valeria Terzi1, Gian Paolo
Ciceri1,
Paolo Provero2,Caterina Morcia1, Primetta Faccioli1. 1C.R.A., Istituto
Sperimentale per la
Cerealicoltura, Via S. Protaso 302, I-29017 Fiorenzuola d’Arda (PC),
Italy and 2Dipartimento
di Genetica, Biologia e Biochimica, Università di Torino, Via
Santena 5bis,
I-10100 Torino, Italy Email:
v.terzi@iol.it Advanced gene
expression analysis
methods, such as microarray and Real Time PCR, as well as more
traditional
ones, such as Northern blotting, require efficient normalization
approaches to
be really informative. Therefore, expression results are usually
normalized
against a control gene that should be expressed in an unchanging
fashion
regardless of experimental conditions (i.e. tissue types, developmental
stages,
sample treatments). However the utilization of single housekeepers
can’t assure
a not-biased result: as a consequence new normalization methods
employing
multiple housekeeping genes and normalizing using their mean expression
have
been proposed, but finding a good set of reference genes is for sure a
non
trivial problem requiring quite a lot of lab-based experimental
testing. In
this work (Faccioli et al, DOI 10.1007/s11103-006-9116-9) , a novel,
agile
approach, based on in silico analysis of plant transcriptome, has been
developed, for the rapid identification of candidate reference genes to
be used
as endogenous control in quantitative gene expression analysis. The
fundamentals of the analysis is based on the fact that housekeeping
genes are
expressed across a variety of tissues and biological conditions, as a
consequence if we select those transcripts that are present in a
remarkable
number of different cDNA libraries we should be able to more easily
identify
potentially constitutive genes to be used as reference genes in
normalization
of expression data. The TIGR Gene Indices
(http://www.tigr.org/tdb/tgi/),
examples of public EST-based species-specific databases, have been used
to
provide such information. Moreover, a web search engine optimization
has been
set up and applied to collect the most interesting information on the
expression profiling of candidate housekeepers on specific experimental
conditions. As a validation test for the method, a qPCR analysis has
been
carried out to verify the expression profile of the selected genes in
different
barley organs and developmental stages and on stress conditions. CAmpER
- A software for the calculation of
amplification efficiencies for real-time PCR-experiments. Blom
J.,
Rückert, C., Kalinowski, J.,
Goesmann, A. Center for
Biotechnology, Email:
jblom@cebitec.uni-bielefeld.de Quantitative
real-time PCR has
become one of the fundamental methods of modern molecular biology and
medicine
for the measurement of gene expression. For the analysis of a real-time
PCR
experiment it is crucial to have exact knowledge of the underlying PCR
kinetics. Especially the amplification efficiencies of an experiment
are of
importance for the analysis of gene expression ratios. For this reason
the
software CAmpER was designed as a software-tool for the automatic
analysis,
annotation, and storage of real-time PCR experiments performed with
different
real-time PCR systems, currently the Lightcycler® (Roche
Diagnostics) and the Opticon® (Bio-Rad Laboratories,
Inc.). CAmpER
obtains the experiment data
by automatically importing the fluorescence and melting-curve data from
the
output files of the real-time PCR-system. Based on the imported
fluorescence
data the amplification efficiencies are calculated for single samples
by two
different independent algorithms. The first algorithm is based on the
DART-PCR
method1, the second algorithm is based on a four parametric
logistic
model2 of the fluorescence curve. Both algorithms were
revised and
improved, especially the DART-algorithm, now calculating precise
crossing
points, which were integer in the original version. The
calculated amplification
efficiencies are used to calculate efficiency-corrected crossing
points. These
corrected crossing points can be used to estimate gene expression
ratios for
all samples, which are more accurate than gene expression ratios
calculated
from uncorrected crossing points. Another
function of CAmpER is the
consistent storage of all relevant information of a real-time PCR
experiment,
giving CAmpER a basic LIMS functionality. It allows users to annotate
the
samples used in the experiment as well as the experimental parameters
in a
standardized format. The annotation information and all other
experiment data
is stored using a relational database management system (here MySQL).
The
modular design of CAmpER allows for extension of the software, for
example to
support additional real-time PCR systems in the future. To ensure
easy access to stored data
and easy use of the implemented functions a convenient user interface
was
designed. This user interface is implemented as a web-based frontend,
making a
local installation of CAmpER needless. Session:
RDML
discussion forum
Chair: J. Hellemans Lecture
hall HS 15 RDML:
real-time PCR data markup language. Vandesompele
Jo, Hellemans Jan Center for
Medical Genetics, Email:
joke.vandesompele@ugent.be 1.
Introduction It is
currently very difficult to
share qPCR data between different laboratories, or exchange data
between
different software packages or analysis tools. The problem is founded
in the
data collection software packages that, depending on the instrument
company,
export information in various file formats (.csv, .txt, .xls), with
different
layout and data field terminology. A common universal format would
allow easy
exchange of raw annotated data and analysis settings for qPCR
experiments. This
would make it possible to include qPCR data and analysis settings in
scientific
papers, allowing both reviewers and readers to re-analyse the data,
much like
the MIAME guidelines propose for microarray experiments. In principle,
the
universal data format should contain sufficient information to
understand the
experimental setup, re-analyse the data and interpret the results. This
data
format could be part of yet to be established MIARE guidelines (Minimal
Information About Real-time Experiments). 2. Discussion We have
drafted a proposal for a
universal data format for the exchange of real-time PCR data, termed
RDML
(Real-time PCR Data Markup Language - http://medgen.ugent.be/rdml/). We
propose
to use an XML-based file type because this format is independent of
computer
hardware, operating system or available software package, and can be
extended
in the future to include additional information if required. The aim of
this
session is to discuss and finalize the RDML specifications. Points to
discuss
include: 1. What
information must be
included? 2. Which
fields are required and
which are optional? 3. Which
fields are free text, and
which have predefined values? 4. What
terminology should be used? 5. How to
obtain broad acceptance of
this standard? 6. Is there a
need for an RDML
working group to continue the development of RDML? Session: New Diagnostic applications with qPCR Chair:
S.
Bustin / K.
Stanley Lecture
hall HS 14 Analysis
of expression signatures associated with
microvascular invasion in colorectal cancer. Rebecca E
Hands 1, Keith
Stanley 2, Sina Dorudi 1, Stephen A Bustin
1 (1) Queen
Mary University of London,
United Kingdom (2) AusDiagnostics, Email:
s.a.bustin@qmul.ac.uk The
development of molecular
analytical techniques and their application to clinical diagnostic
studies has
been rapid. However, the reliable interpretation of results obtained
from gene
expression analyses is critically influenced by the selection of
appropriate
study material. Colorectal cancer biopsies contain multiple cell types,
are
multiclonal and are characterised by distinctive histopathologies with
discrete
genetic and gene expression profiles. As a result, gene expression
profiles
from bulk biopsies are likely to obscure clinically relevant expression
signatures from areas associated with poor treatment outcome, such as
those
characterised by vascular and lymphocytic infiltration. A critical
consequence
of this heterogeneity is that such samples are unable to provide robust
expression signatures capable of accurate prognostic stratification of
individual colorectal cancer patients following surgery. Our solution
involves
the establishment of a tumour atlas for clinically relevant
histopathological
areas in colorectal cancers. We achieve this by using laser capture
microdissection in combination with multiple tandem RT-qPCR to
delineate
distinct gene expression features associated with high metastatic
potential and
poor treatment outcome. Use
of Tomato Mosaic Virus (ToMV) as Internal Positive
Control (IPC) in different RT-PCR settings. Helga
Mairhofer1, Martin
Obermeier1, Günter Adam2, Hans Nitschko1 (1) Max von
Pettenkofer-Institute,
Ludwig-Maximilians-University Munich, Department of Virology, Munich,
Germany
(2) Pflanzenschutzamt Hamburg, Biozentrum Klein Flottbek, University of
Hamburg, Hamburg, Germany Email:
nitschko@mvp.uni-muenchen.de Objectives:
The use of an Internal
Positive Control (IPC) in each individual specimen is highly
recommended and
increasingly practiced in routine clinical virus diagnostics. We used
Tomato
Mosaic Virus (ToMV), a highly stable single-stranded plant RNA virus,
to
monitor the efficacy of nucleic acid isolation, reverse transcription
and
amplification in different RT-PCR settings. Methods: A
short 190 nt sequence
located in the coat protein region was coamplified with ToMV- specific
primers
after addition of ToMV-IPC directly into the clinical specimen prior to
extraction.. We used different nucleic acid isolation systems to
extract viral
and IPC RNA: Roche, MagNAPure, Roche AmpliPrep and the High Pure Viral
Nucleic
Acid Isolation Kit as a manual extraction protocol. The isolated RNAs
were
investigated using different amplification and detection platforms:
Roche
LightCycler 2.0, ABI TaqMan 7500 fast and conventional RT-PCR with
visualisation of the amplicons by agarose gel electrophoresis. The
concentration of the added ToMV was carefully adjusted to ensure
reliable and
reproducible detection without significant loss of sensitivity
especially for
weak positive RNA signals from respective viruses to be detected in the
patient
specimen. We optimized the protocol by evaluation different
primer/probe
concentrations for reverse transcription and RT-PCR amplification using
a
variety of difficult clinical specimen such as stool, BAL, biopsies,
pharyngeal
lavages, swabs, urine and ENTA but also peripheral leucocytes, plasma
and
serum. Norovirus positive stool samples and influenza A virus (H5N1)
positive
materials were applied to analyze in detail the performance of the
spiked,
co-amplified IPC as well as the amplification of the specific viral
RNAs. Results: More
than 300 different
clinical specimen were analysed. The IPC could be reliably and
repeatably
detected in most of the samples. In some specimen inhibition was
clearly
demonstrated as indenpendently of the extraction/amplification protocol
no or
only weak IPC signals were detectable resulting in a shift of Ct-values
and/or
flattened amplification curves. Upon ten-fold dilution IPC and virus
specific
RNA-signals could be recovered with appropriate signal intensity. We
could also
identify samples with insufficient efficacies in either RNA extraction,
reverse
transcription and/or amplification (below 5% of all samples), clearly
depending
on the type of sample material, extraction method and amplification
conditions.
As expected not all isolation/amplification methods are equally well
suited to
guarantee optimal performance. Conclusions:
Tomato Mosaic Virus is
a stable, reliable and easy to handle plant virus which can be isolated
in high
concentrations, stored unendangered and used safely as an Internal
Positive
Control in RT-PCR detection protocols for human RNA viruses in clinical
specimen. Evaluation
of endogenous control genes for real-time
quantitative PCR in breast cancer tissues. McNeill R.E., Miller, N.
and Kerin, M.J. Department of
Surgery, Clinical
Science Institute, National Email:
roisin.mcneill@nuigalway.ie Quantitative
real-time PCR (qPCR),
one of the most sensitive and specific quantitative methods for gene
expression
analysis has become the basis of many breast cancer biomarker studies
as well
as novel diagnostic and prognostic assays. Normalisation of relative
qPCR data
is required to control for variation introduced during sample
preparation,
particularly to control for differences in the quantity and quality of
RNA used
in each reaction. Endogenous control (EC) genes, used to normalise
relative
qPCR data, should ideally be expressed constitutively and uniformly
across
treatments in all test samples. The aim of this study was to identify
the most
suitable endogenous control gene(s) from a panel of twelve candidates (
GAPDH , ACTB , TFRC , ABL , PPIA
, HPRT , RPLP0 , B2M , GUSB ,
MRPL19 , PUM1 and PSMC4 )
for the quantification of gene expression by relative comparative qPCR
in
breast cancer tissues using the oestrogen receptor alpha ( ESR1 )
transcript as a target gene. Primary
breast tumour tissues,
malignant (n=21) and benign (n=8) were obtained from consenting
patients during
primary curative resection in There was a
significant difference
in candidate EC variability within group (P<0.01). GeNorm and
Normfinder
identified the same two genes as most stable. GAPDH was less stable
than either
of the two genes identified. ESR1 expression was estimated to
be
appreciably higher in malignant tissues than in benign tissues
irrespective of
which EC was used. In conclusion, two genes have been identified as
good
candidate ECs for the normalisation of qPCR gene expression data in
these
tissues. 1. Vandesompele,
J.; De Preter, K.; Pattyn, F.; Poppe, B.; Van Roy, N.; De
Paepe, A.; Speleman, F. Genome Biol 2002, 3, (7), RESEARCH0034. 2. Andersen,
C. L.; Jensen, J. L.; Orntoft, T. F.
Cancer Res 2004, 64, (15), 5245-50 Fluorogenic
Quantitative PCR for Non-laboratory
Applications. Lee M.1, Squirrell D.1,
and Wakeley, P.2 1Enigma
Diagnostics Ltd, Building
224, Tetricus Science Park, Dstl Porton Down, Salisbury, Wiltshire SP4
0JQ and 2Veterinary
Laboratory Agency, Technology Transfer Unit, Biotechnology, New Haw,
Addlestone, Surrey KT15 3NB Email:
martin.lee@enigmadiagnostics.com For several
years both defence and
homeland security organisations have used QPCR as a powerful tool for
the
detection of bacterial and viral biological agents. Field laboratories
using
commercial thermal cyclers and nucleic acid extraction systems have
been
deployed, but skilled operators and an ensemble of consumables were
required.
Enigma Diagnostics has developed a better solution. The Enigma FL is a
portable
device that can be operated after ten minutes of training. It carries
out
automated extraction, amplification and analysis with the sample loaded
into a
single cartridge containing all of the reagents, including a
freeze-dried PCR
mix. The device can be operated from mains, battery or a vehicular
power supply
opening up numerous new applications for nucleic acid testing outside
of the
laboratory. Here we present a detailed technical overview of the
instrument and
technology within, whilst illustrating the utility of the device for
the
veterinary testing for a reportable infectious disease, bovine viral
diarrhoea
virus (BVDV), in a UK-based field trial. Real-time
PCR in Diagnostic Microbiology - a review on
9 years of R&D in an academic environment. University Email:
udo.reischl@klinik.uni-r.de For more than
a century, pathogenic
microbial detection and identification have relied on the ability to
cultivate
organisms in the diagnostic laboratory. About twenty years ago a
practical
method of amplifying DNA for the clinical laboratory appeared - well
known
today as the PCR. Application of nucleic acid amplification,
distinguished by
its high analytical sensitivity and specificity, circumvent the
necessity for
microorganisms to be isolated in pure culture prior to their definitive
identification. In the early 90's many of us played around with block
cycler
PCR devices, special brands of thermostable polymerases and
semiquantitative
amplicon detection in agarose gel analysis. But the true quantum leap
in
technology was associated with the event of real-time PCR - where
detection of
amplicons and monitoring of the amplification process was possible
after each
thermocycle. With the introduction of the LightCycler instrument in
August
1998, the advantages of real-time PCR were accessible even for routine
laboratories and molecular diagnostics become particularly popular.
Combining
PCR amplification and probe detection of target nucleic acids in the
same
closed reaction vessel permits dynamic assessment of PCR amplicons,
quantification of target organisms and concurrently minimizes the
possibility
of contamination by exogeneous nucleic acid in the workflow of
molecular
microbiology. Due to the widespread application of real-time PCR and
the
development of various assay formats, a huge spectrum of diagnostic
in-house
protocols or commercial kits are now available to detect almost any
bacterial
or fungal organsim as well as related pathogenicity factors. The
versatility of
this approach is exemplified by applications which identify human
pathogens,
relevant mutations or antimicrobial resistance genes. Moreover, a
number of
automated nucleic acid extraction systems will be discussed which have
been
developed to standardize and economize the diagnostic workflow from
receipt of
a clinical specimen to reporting the corresponding PCR result. Although
the
present technology can be considered to be robust and reliable for
routine
purposes, further developments are ahead - and high resolution melting,
multiplexing or high-throughput testing in microwell formats will
definitely
expand the spectrum of diagnostic applications in the months or years
to come. Multiplex
quantitative PCR for detection of Ehrlichia
canis, Babesia canis and canine ACTB gene. Ofer Peleg1, Gad Baneth2,
and Shimon Harrus2 1Zotal LTD, Email:
oferp@zotal.co.il The ricketsia
Ehrlichia canis and
the protozoon Babesia canis vogeli share the Ixodid tick Rhipicephalus
sanguineus as a vector. Canine monocytic ehrlichiosis is acknowledged
as an
important and potentially fatal infectious disease of dogs and other
members of
the Canidae family, while parasitic Babesia species infect
erythrocytes,
causing anemia in the host. As a result of their common vector,
concurrent
infections with both pathogens are common. Therefore, in this study we
attempted to construct a multiplex quantitative PCR assay for the
detection of
both pathogens simultaneously. Using the
qPCR Singleplexer and
Multiplexer software (GenAphora) for singleplex and multiplex primer
and probe
design, Singleplex qPCR assays for detection of the E. canis-16S rRNA
gene,
Babesia vogeli-hsp70 gene and the canine beta-Actin (ACTB) as a
reference gene
were constructed. In addition, a multiplex assay for the simultaneous
detection
of all three genes in a single tube was constructed. The sensitivity of
the
multiplex was reduced compared to the singleplex assays. The singleplex
assays
could demonstrate detection of 1-10 molecules of organism (DNA
copies)/µl. while
the multiplex assays were able to detect ~100 molecules of organism
(DNA
copies)/µl. The probable causes for this reduction are system
resources
deficiency (e.g. nucleotide and ion deficiency) due to the occurrence
of 3
simultaneous reactions, and the presence of 3 different primer
probe-sets in
the same tube in addition to the presence of genomic-DNA and blood DNA
extraction byproducts in the reaction tube. Of these, the major
contributor for
the multiplex sensitivity reduction was the existence of several
reactions in
the same tube. Improvement in the sensitivities of the multiplex test
is
currently underway. The present
assay enables the
quantitative detection of two common canine blood pathogens
simultaneously. It
can thus indicate the institution of proper treatment and monitoring
the
response to treatment with the changes in pathogen numbers in the blood. Children's
Cancer Research
Institute, Email:
sandra.preuner@ccri.at Rapid and
sensitive detection of
invasive fungal infections by a 2-step
pan-fungal real-time PCR
assay S. Preuner,
T. Lion, Children´s
Cancer Research Institute, Invasive
fungal infections (IFI) are
a major cause of life-threatening situations in immunocompromised
patients
(bone marrow transplant patients, patients with haematological
malignancies,
HIV patients). The incidence of IFI in these patients is estimated at
10-40% .
The most common organisms involved are Aspergillus and Candida species
(>
90%), with a mortality rate of 10-40%. The introduction of new
antifungal and
antineoplastic treatment results in changes in the epidemiology of
fungal
infections. The so-called “emerging fungi” including e.g. Fusarium
spp.,
Mallassezia spp., Trichosporon spp., Cryptococcus spp. play an
increasing role.
Thus early, sensitive and broad detection of fungi has to be
implemented.
Currently used detection methods, such as microbiological cultivation,
serological tests, histological techniques or imaging methods are
mostly
inconsistent in sensitivity and specificity or are time consuming.
Therefore
antifungal treatment is often empirical, resulting in unnecessary
treatment of
patients in absence of IFI or in delay of treatment in the presence of
IFI. To
provide a reliable tool for diagnosis of IFI, we have developed a
two-step
pan-fungal real-time assay targeting the 28s rRNA gene. Assay I
(“Mould-Assay”)
covers all clinically relevant Aspergillus species and a number of
other
important moulds. Assay II (“Yeast Assay”) detects all Candida species,
Trichosporon spp., Cryptococcus spp, and a number of filamentous fungi
not
covered by the Mould Assay. In total, more than 80 different fungal
species,
including also a large panel of “emerging fungi” can be detected and
quantified. High homology with human 28S rRNA gene required the
employment of
LNA (locked nucleid acid) modified primers and probes, resulting in
enhanced
affinity for the complementary sequence. Due to the composition of the
cell
wall, DNA extraction had to be modified by the introduction of
enzymatic and
mechanical pre-treatment. The
occurrence of contaminations by
airborne spores in reagents for DNA extraction and PCR were a major
issue. This
problem was overcome by using molecular grade reagents and the
application of a
closed extraction system, the MagnaPure System ( Rapid,
On-demand Detection of Drug Resistant
Microorganisms by using the GeneXpert. Persing DH. Email:
david.persing@cepheid.com Detection of
drug-resistant
organisms, especially those that cause hospital-acquired infections,
has become
an increasingly important role for the clinical laboratory.
Conventional
culture methods are too slow to be of use in the most effective
hospital
infection control strategies, which require rapid recognition and
cohort
isolation. Molecular methods based on PCR have improved turnaround
time, but
their dependence on batch-mode processing can stretch effective
turnaround
times to 24 hours or more. We have developed rapid cartridge based
tests for
several drug resistant organisms. These tests carry out automated
sample
extraction from crude clinical specimens such as sputum, blood, or
swabs and
use realtime PCR to detect targets and internal process controls that
are
multiplexed in 4 to 6 color channels. Results are delivered in as
little as 30
minutes in order to facilitate high-impact medical management or
treatment
decisions. Because each cartridge contains it own controls, tests can
be run
on-demand, in batches or one at a time, within the random-access
GeneXpert
system. Tests for different analytes, using diferent reagents and
distinct
thermal cycling conditions, can be run side-by side upon arrival in the
lab,
thus keeping effective turnaround time to a minimum. Data will be
presented
from Cepheid's test released for detection of methicillin resistant S.
aureus,
and for tests in development for drug resistant tuberculosis and
influenza
virus. Applications
of high resolution melt curve analysis
for genetic diagnostics. White H.1, Watkins G. 1,
Hall V.1 and Cross NCP. 1 (1) National
Genetics Reference
Laboratory ( Email:
hew@soton.ac.uk High
resolution melt curve analysis
(HRM) is a simple & cost effective post-PCR technique that can be
used for
applications such as high throughput mutation scanning, detection of
somatic
mutations, genotyping & methylation profiling. The technique
requires the
use of standard PCR reagents only and a dsDNA binding dye. NGRL ( We have
evaluated the use of 3
machines for mutation scanning using HRM: RotorGene 6000 (Corbett
Research),
HR-1 and 384 well LightScanner (Idaho Technology). Eleven amplicons
were
analysed which varied in size from 139 - 449bp and had GC contents
ranging from
22 - 79%. The types of mutations analysed included all possible point
mutation
base substitutions and 1 and 2bp insertions and deletions. 624 blinded
samples
were amplified in the presence of the saturating double stranded DNA
binding
dye LCGreen Plus (Idaho Technology) using a RotorGene 6000. Identical
PCR
products were analysed using HRM on the three platforms. The overall
sensitivity and specificities for each machine were 100% & 95%
(RotorGene
6000), 98.4% & 95% (HR-1) and 99% & 88% (LightScanner)
respectively. We
concluded that HRM is an extremely sensitive and specific technique for
mutation scanning that could be easily integrated into clinical
diagnostic
pre-screening strategies. This closed tube method has advantages over
current
mutation scanning techniques since it requires no post-PCR handling
(minimising
the risk of PCR contamination) and no separation step, which improves
analysis
time. The technique can also be used to scan for somatic mutations and
has a
sensitivity of detection of approximately 2%. NGRL ( Huggett J. F. Centre for
Infectious Diseases &
International Health, Email:
j.huggett@ucl.ac.uk Infectious
diseases are a major
cause of mortality and morbidity in the developing world. Tuberculosis
(TB)
causes ~2 million deaths per annum; this is effectively the death toll
attributed to the 9/11 attacks on the World Trade Centre every 12
hours.
Diseases like TB are notoriously difficult to diagnose. We are using
qPCR and
reverse transcriptase qPCR to establish the potential role of molecular
methods
in the management TB and Pneumocystis pneumonia (PCP), two respiratory
infections with similar symptoms. While there are many reports
describing
molecular assessment using PCR for diseases like TB and PCP, the
clinical
utility of these tools has not been uniformly successful and they are
generally
not used as a routine diagnostic method, especially in the developing
world. There are
many reasons why PCR has
not been more successful in diagnosing TB and PCP, but a lack of
consistency in
the literature is a major factor. This is confounded by the frequently
expressed notion that the publications have assessed the diagnostic
efficacy of
PCR. This notion is wrong. These reports are assessing the diagnostic
efficacy
of a multistep process, the final step of which is PCR. Consequently,
for a
report to be thorough there must be detailed explanation of chosen
sample,
storage and extraction procedure. Molecular assessment by PCR (or other
enzymatic amplification methods) should also control for inhibition.
Finally
the PCR reaction used to detect the pathogen of interest should have a
defined
reproducibility, detection sensitivity and ideally efficiency. We are
approaching this problem by
using in silico methods to assist the assay design and internal
controls to
provide confidence in our results. Concomitantly we consider different
storage
and extraction procedures from a range of clinical samples for optimal
nucleic
acid recovery and removal of inhibitors. We are also investigating the
use of
novel clinical samples including urine to target pathogen transrenal
DNA as a
tool for monitoring respiratory infection. Our infectious disease
diagnostic
and prognostic studies using real time PCR can provide valuable
clinical
information that will be important in the management of infectious
diseases in
the developing world. Two
new probes for Real-time PCR: EasyBeacons™ and
HydrolEasy™ probes. Christensen U. PentaBase,
Denmark Email:
ubc@pentabase.com We would like
to introduce two new
probe systems for real-time detection during PCR amplification:
EasyBeacons™
and HydrolEasy™ probes. Both type of probes are modified with the DNA
analogue
Intercalating Nucleic Acid (INA®) or INA®-like
molecules
and labelled with a fluorophore and a quencher. The fluorophores are
compatible
with commercially available real-time PCR instruments and hence the
technology
can be used on standard equipment. The novel
features of EasyBeacons™
and HydrolEasy™ probes are introduced by INA®. Common
for the two
probe systems are, that they are easy to design and very specific. INA®-modifications
increase binding to their complementary targets with as much as up to
11°C per
modification. They have an inherent quenching mechanism, yielding low
background fluorescence from unbound probes. This quenching mechanism
is
independent of temperature, making multiplexing easier. EasyBeacons™
are nuclease resistant
probes that can be used both real-time during the amplification
reaction and in
end-point detections. This dual detection mechanism is making
EasyBeacons™
ideal for detection of SNPs, MeC patterns and other variations, as the
real-time result can be verified by an end-point affinity measurement,
similar
to the one used with non-specific dyes like SYBR® green.
We will
also present fast optimisation protocols for using EasyBeacons™.
Examples shown
will include the detection of methylation pattern of single CpG duplets
as well
as detection of challenging SNPs. HydrolEasy™
probes are hydrolysed
during the amplification reaction, cleaving the fluorophore from the
rest of
the probe. Besides being high affinty probes, due to the INA®
modifications, the unbound probes have a low background fluorescence
due to
hydrophobic interactions between INA® modifications.
HydrolEasy™
probes are ideal for two-step real-time PCRs. Susanne
Füssel1, Susanne
Unversucht1, Andrea Lohse1, Silke Tomasetti1,
Anne-Katrin Rost2, Manfred P. Wirth1, Axel Meye1,
Thomas Köhler2 1Dept. of
Urology, Technical Email:
susanne.fuessel@uniklinikum-dresden.de In this study
the novel real time
PCR detection format “TripleHyb” was optimized for the detection of
single
nucleotide polymorphisms (SNP) for the known C-460T polymorphism of the
VEGF promoter.
“T” instead of “C” at position -460 is believed to be associated with
altered
VEGF promoter activity possibly leading to increased incidence of
different
diseases and malignancies such as prostate cancer. Briefly, the
TripleHyb format
besides a conventional set of primers comprises a pair of
oligonucleotide
probes which consist of a target-complementary subsequence and a
target-unrelated subsequence for the formation of a stabilizing stem
structure
between the probes. The labeled upstream probe is cleaved by
5`-exonuclease
function of taq polymerase as long as full complementary with target is
achieved, whereas the probe is displaced if at least one mismatch is
present
resulting in lack of fluorescence signal. To find out at which
position(s) of the
upstream probe a mismatch causes SNP selectivity a common VEGF promoter
specific set of primers and 5 different sets of either complete target
matching
or single mutated fluorescence labeled upstream and the corresponding
non-labeled downstream probes were developed. The mismatches were
located
either at positions 1, 2, 3, 4, or 5 (probe set 1-5) of the upstream
probe`s
5`-end. Optimizations of PCR conditions using a plasmid containing a
wild-type
or C460T polymorphism subsequence were performed. A sensitive detection
of the
specific genotype was feasible using probe sets 3, 4 and 5,
respectively
without need for running subsequent melting curve analysis. When
applying a
1:1-mixture of both wildtype and mutated template variants a
reproducible and
reliable discrimination between the SNP variants was possible with all
three
systems. We conclude that with TripleHyb a broad hot spot region within
the
upstream probe may be exploited thus simplifying the probe design. Set 3 was
selected to analyze 80 DNA
samples from prostate cancer patient for the respective mutation
frequency. The
clinical data are still in the evaluation process. Allergen
determination in food by multiplex qPCR. Köppel R. Kantonales
Labor Email:
rene.koeppel@klzh.ch The cantonal
laboratory examines
food samples for safety and fraud. Two to six percent of the human
population
exhibit allergic reactions due to the consumption of food containing
allergens.
To increase the well being and safety of these people food samples are
regularly examined for the presence of allergens and the compliance of
declaration. Currently there are 18 allergens listed by the food law of
Europe
and Quantitative
DNA Methylation Analysis. Serena Vinci,
Francesca Malentacchi, Roberta Cascella,
Francesca Salvianti, Mario Pazzagli, Pamela Pinzani, Claudio Orlando. Clinical
Biochemistry Unit,
Department of Clinical Physiopathology, Email:
c.orlando@dfc.unifi.it Genome
stability and normal gene
expression are maintained by a constant and predetermined pattern of
DNA
methylation. ‘Epigenetics’ is defined as heritable changes in gene
expression
that do not result from alteration in the gene nucleotide sequence.
These
include DNA methylation and histone modifications, that provide an
"extra" layer of transcriptional control. Epigenetic abnormalities
have been found to be causative factors in cancer, genetic disorders
and
pediatric syndromes as well as contributing factors in autoimmune
diseases and
aging. The detection
of abnormal patterns
of DNA methylation, beside the relevant insights in the comprehension
of
molecular mechanisms of these diseases, seems to provide a new and
promising
biomarker for identifying occult circulating DNA inside complex
biological
matrices (blood, urine, sputum, stool, etc.) for early cancer
detection. It
should be noted that changes in DNA methylation also occur in normal
epithelia
in physiological and pathological conditions. Thus, conventional
qualitative
techniques for methylated DNA sequence cannot guarantee the expected
clinical
relevance for routine applications. Quantitative Methylation Specific
PCR
(QMSP) is capable of detecting methylated alleles in the presence of
10000-fold
excess of unmethylated alleles and in addition it can provide a
quantitative
estimation of the number of abnormally methylated sequences in clinical
samples. In our study
QMSP was applied to
determine aberrant promoter methylation profile of the 5’ region of 3
apoptosis-associated genes (Bcl2, hTERT e DAPK) in urine sediments as
potential
biomarkers for bladder cancer detection. DNA hypermethylation was
detected in
about 80% of bladder cancer patients, with a specificity of 91%.
Methylation
levels were variably but significantly associated with clinical and
pathological markers. Another
interesting field of
application of quantitative measurement of occult circulating DNA is
represented by the non-invasive diagnosis of some pregnancy-associated
diseases, such as pre-eclampsia. Up to now, the lack of a fetal DNA
marker that
can be universally detected in maternal plasma has limited the clinical
application of this technology. It is now evident that epigenetic
differences
between the placenta and maternal blood cells could be used to detect
fetal DNA
in maternal plasma. Maspin gene promoter is heavily methylated in
maternal
blood cells and hypomethylated in the placenta. Placental-derived
hypomethylated maspin is detectable in maternal plasma regardless of
the fetal
sex and genetic variations and thus could serve as a universal fetal
DNA
marker. Recently it was demonstrated that the promoter of the RASSF1A
tumor
suppressor gene is hypermethylated in the placenta but hypomethylated
in
maternal blood cells, a methylation pattern that is exactly opposite to
maspin
promoter. We explored both these strategies to set-up a fetal DNA
marker easily
detectable and quantifiable by real-time PCR. Validation
of StaRT-PCR for reliable and robust
analysis of variably degraded formalin-fixed paraffin-embedded or fine
needle
aspirate biopsy samples. James C.
Willey1, Charles
Knight2, Bradley Austermiller2, Thomas Blomquist1,
Erin Crawford1, (1)
University of Toledo, United
States of America (2) Gene Express, Inc. Email:
james.willey2@utoledo.edu It is widely
anticipated that a
transcript abundance (TA) measurement method that generates data
suitable for
regulatory review will facilitate development of new drugs and improved
diagnostic tests. A particular challenge is to maintain these
performance
characteristics using clinical samples that often unavoidably are
degraded to
different degrees. This study was an effort to assess the utility of
Standardized RT-PCR (StaRT-PCR™) for reliable and robust analysis of
formalin
fixed paraffin embedded (FFPE) or transthoracic fine needle aspirate
(FNA)
biopsy samples. Seven genes were assessed relative to the ACTB loading
control
gene, including four genes reported to be associated with resistance to
pemetrexed and three genes that accurately diagnose bronchogenic
carcinoma,
MYC, E2F1, and CDKN1A. A 3’, middle, and 5’ region of the transcript
was
assessed for each gene. Each region was selected for analysis based on
thermodynamic analysis to identify short sequence regions most
resistant to
degradation. Cell culture populations from each of seven carcinoma cell
lines
were split into two samples, one of which was pelleted and snap frozen
(FF),
the other pelleted, formalin fixed, and paraffin embedded (FFPE). RNA
was
extracted from each sample, DNase treated, quantified, and reverse
transcribed
using multiple different RT priming methods. Genomic DNA was assessed
and
ensured to be negligible. TA for each gene was measured relative to a
known
quantity of its respective internal standard within a standardized
mixture of
internal standards (SMIS<sup>TM</sub>). This provided the
performance characteristics recommended by the FDA to generate data
suitable
for regulatory review, including intrinsic quality control to prevent
false
negatives and false positives and to control for effects of interfering
substances such as gene-specific inhibitors. The integrity of each RNA
sample
was assessed according to yield of ACTB cDNA molecules/ng RNA. For the
seven
FFPE samples, the ACTB cDNA yield ranged from 0.3% to 1.4% of that
obtained
from the respective matched FF sample. In spite of extreme degradation,
TA
value for each target gene normalized to ACTB was highly correlated for
each
FF/FFPE pair. However, the correlation was least for the two FFPE
samples with
the lowest ACTB cDNA yield from RT, each with 420 ACTB molecules/ng
RNA.
Primers spanning the most 5’region of each transcript yielded results
best
correlated between FF and FFPE. Spontaneous priming was as efficient as
oligodT
or gene specific priming and also yielded reliable FF/FFPE results.
Based on
this study, FFPE or other clinical samples will be judged suitable for
analysis
if RT yields more than 1,000 ACTB cDNA molecules/ng RNA. The StaRT-PCR
reagents
validated in this study will be used in clinical trials. J.C. Willey
has equity
interest and is consultant for Gene Express, Inc. E.L.Crawford has
equity
interest in Gene Express, Inc. Design
and validation of a robust diagnostic assay
(prv-1 gene) based on real-time RT-PCR. Häusler
P., Bohle
V. Kooperationsgemeinschaft
molekulare Labordiagnostik, Germany Email:
phausler@oncoscreen.com Quantitative
PCR of the prv-1 mRNA
was set up and validated as a robust and reliable method for clinical
routine
diagnostics of Polycythemia vera rubra (PV) on the basis of peripheral
blood
specimens. Initially, the conditions of blood storage and shipping were
evaluated. Then an optimised technique for cDNA preparation from
granulocytes
was established, followed by the identification of a panel of three
reference
genes out of a panel of six candidate reference genes, among them genes
that
had been suggested previously for hematologic diseases by the Europe
Against
Cancer group (EAC). This primary selection identified beta actin and
G6PD as
optimum reference genes that are clearly superior to B2M, GUS or ABL.
The
ranking of GAPDH remains controversial and depends on the mathematical
model
used for evaluation. On the basis of subsequent stability data where
blood was
stored at diverse ambient temperature the expression GAPDH proved to be
rather
unstable upon storage. On the basis of these data an assay was designed
and
validated. Limit of detection, limit of quantification, intra assay
variability
and inter assay variability were evaluated, respectively. Finally, a
schedule
for the collection of long term stability data was set up. This assay
now needs
to be clinically validated in a prospective trial. The modus operandi
outlined
here we suggest as a standard procedure for designing diagnostic assays
based
on quantitative real-time PCR. Assessment
of yeast intron insertion on expression and
mRNA level of the human alpha-1 Antitrypsin cDNA in Pichia pastoris. Hasannia S.1, Lotfi A. S.2,
Mahboodi F.3 and Mohsenifar A.4 Email:
s_hasannia@guilan.ac.ir Human Alpha-1
antitrypsin (AAT) is a
Serpin (serine Protease inhibitor) and an inhibitor of neutrophil
elastase.
This glycoprotein consists of 12% carbohydrate, 394 amino acids and has
a
molecular weight of 54 KD. As this protein is glycosylated and has a
high MW.
Any procedure which can increase the expression and secretion of the
recombinant protein in yeast can be a useful in the production of this
medicine. Although various methods, such as using various promoters,
selection
of specific untranslated region (UTRs), various signal sequences,
appropriate
inducer various mutants and other biotechnological procedures are being
employed to increase recombinant protein expression in yeast. In this
study a
new technique which concentrates upon regulation in the nucleus and
uses yeast
intron integration in the human AAT cDNA, has been used to study its
effect on
gene expression. There are various report indicating a role for intron
sequence
in the expression of homologous and heterologous protein expression in
animal
and plant cells. Increased recombinant protein expression due to
introns is
termed intron-mediated enhancement or IME. In this
study, the yeast Pichia
(strain GS-115) and the integrating shuttle vector pHIL-S1 has been
used. At
first, the pHIL-S1 vector carrying AAT was introduced into the yeast
using the
chemical – electroporation method and the resultant strain named
GS-AAT. In
order to integrate the yeast intron in a suitable site in the AAT cDNA,
segment
of exon II, yeast intron and multi-exon segments III, IV and V were
separately
amplified using specific primers for PCR and then these sequences were
inserted
into the bacterial vector pBluescript KS. Then the cDNA carrying the
intron was
separated from the vector and integrated in the pHOL-S1 vector and
subsequently
transferred to the yeast (strain GS-AATint). The two strains of yeast
obtained
were cultured in the same conditions and in media specific using
SDS-PAGE and
TIC, which is highly specific for assessment of inhibitory activity of
AAT. In
order to measure the cytoplasmic concentration of the related mRNAs in
the two
strains, Pfaffl method (relative quantitative RT-PCR) with SYBR green
were
used. Recombinant protein expression in both strains could be assessed
after
the second day and this indicates the yeast had carried out the
splicing
correctly. A comparison of the expression at 72 hrs in two media using
the
transformed yeast with TIC showed a 23 fold increase in the strain
carrying the
intron relative to the strain not carrying the intron. On the other
hand,
results obtained from RT-PCR show that the concentration of the
cytoplasmic
mRNA carrying the intron has increased 35 fold. The results obtained
showed
that integration of yeast intron in human cDNA in yeast can cause an
increase
in expression and subsequently secretion and can be used as a useful
technique
producing recombinant glycoproteins in Pichia pastoris. High
Sensitivity QuantiGene 2.0 Assay for Direct
Quantification of mRNA Transcripts. Son Bui, Nina
Nguyen, Yunqing Ma,
Quan Nguyen, George Zheng, Jessie Wu, Wen Yang, Botoul Maqsodi, Joan
Davies,
Jason Li, Gary McMaster, Frank Witney, Yuling Luo Panomics,
Inc., Email:
yluo@panomics.com QuantiGene
assay is a branched DNA
signal amplification technology that quantitatively measures mRNA
directly in
cell lysates and tissue homogenates without the need for RNA isolation,
reverse
transcription, or target amplification. Although the assay has
demonstrated the
best precision and accuracy among all platforms evaluated in the MAQC
(MicroArray Quality Control) study, the assay detection sensitivity is
around
6,000 copies, limiting its application in low copy mRNA detection
situations.
We now report the development of QuantiGene 2.0. The assay maintains
the same
level of precision and accuracy as QuantiGene, but with increased
detection
sensitivity now below 200 copies for every gene tested. QuantiGene 2.0
has been
used in quantifying mRNA in purified total RNA, cell lysates, and
tissue
homogenates. Furthermore, QuantiGene2.0 has proven to be well suited
for mRNA
quantification in FFPE (Formalin-Fixed and Paraffin Embedded) and whole
blood
samples with exceptionally high sensitivity. Side-by-side comparison of
assay
performance demonstrated that the assay sensitivity of QuantiGene 2.0
is now
comparable to, and in many cases, better than qPCR. Tracing
the source of faecal pollution in water by
qPCR: quantitative microbial source tracking (QMST). Georg H.
Reischer1, David C.
Kasper1, Ralf Steinborn2, Robert L. Mach1,
Andreas H. Farnleitner1 1Institute for
Chemical Engineering,
Gene Technology Group, Vienna University of Technology, Vienna, Austria
and 2Institute
of Animal Breeding and Genetics, Department for Animal Breeding and
Reproduction,
University of Veterinary Medicine, Vienna, Austria Email:
reischer@mail.zserv.tuwien.ac.at One special
field of application for
qPCR that has only recently been developing is quantitative microbial
source
tracking (QMST) in water. QMST allows the allocation of faecal
pollution in
water to a specific source group e.g. human or animal sources by
detection of
source-specific faecal microorganisms. This information is vital for
target-oriented measures for remediation of faecal influence on waters
resources
used for various purposes such as drinking water abstraction or
recreation. The
two most crucial requirements for such methods are the
source-specificity
(presence of the marker target in the faeces of one group, absence in
all other
faecal sources) and the sensitivity (high prevalence and abundance of
the
target organism in the specific source, sensitivity of the detection
method). In
order to meet those requirements we recently developed two qPCR methods
for the
source specific detection and quantification of two QMST markers
specific for
human (BacH) and ruminant (BacR) faecal input, respectively. The
methods were
developed for application in an alpine karst spring catchment area. The
low
levels of faecal contamination present in this environment demanded
detection
tools with a sensitivity way beyond the hitherto available methods. The
assays
detect faecal 16S rRNA gene markers from the bacterial phylum Bacteroidetes
using
TaqMan MGB probes. The qualitative and quantitative detection limits of
the
assays were 6 and 30 marker copies respectively. Source-specificity was
found
to be very high when applying the methods on hundreds of faecal DNA
extracts
from various sources from Silvia Calatroni, Barbara Rocca,
Ilaria Giardini, Marina Boni, Irene Dambruoso, Paolo Tarantino, Paolo
Bernasconi Division of
Hematology - IRCCS Policlinico
S. Matteo Foundation, Email:
s.calatroni@smatteo.pv.it The molecular
signature of BCR-ABL
fusion gene in chronic myeloid leukemia (CML) provides a unique tool
for
diagnosis and monitoring of tumor burden during therapy. The
introduction of
imatinib mesylate, allowing the achievement of high rates of clinical
and
cytogenetic remission, has revolutionized the treatment of CML patients
and
reinforced the fundamental role of BCR-ABL transcript levels monitoring
by
RT-qPCR to assess minimal residual disease. Nevertheless, many
procedural
aspects of this complex technique require a strong inter-laboratory
optimisation and recommendations for harmonizing the different
methodologies
have recently been proposed. The Xpert BCR-ABL MonitorTM,
recently
developed by Cepheid (
Session:
High
throughput quantitative PCR
Chair: N.
Zoric / L. Warren Massively
Parallel, Nanoliter-scale PCR for High
Throughput Genomics. Brenan C. BioTrove
Inc., Email:
cbrenan@biotrove.com There is an
increasing need for
accurate and precise high throughput SNP genotyping and quantitative
analysis
of gene expression in diverse areas as pharmaceutical research and
discovery,
agricultural biotechnology, environmental testing and public health. In
response, BioTrove has developed and is commercializing a nanofluidic
platform
uniquely capable of meeting these emerging requirements by enabling
rapid, high
throughput PCR assays in a nanoliter format. Called the OpenArrayTM,
the device
consists of a rectilinear array of 3072 through-holes in a microscope
slide-sized stainless steel platen. Polymer coatings making the
interior
surface of each hydrophilic and the exterior surface of the platen
hydrophobic
are applied and allow PCR to be performed in each fluidically isolated
33 nL
through-hole. After an overview of the OpenArray technology, a number
of
biomedical applications will be described based on SNP genotyping with
the
Taqman PCR assay and quantitative measurement of gene expression with
real-time
SYBR Green PCR. Towards
High-Throughput Single-Cell Expression
Analysis. Warren L. Email:
luigiw@stanford.edu Understanding
the developmental
program by which stem cells differentiate into diverse cell types is a
central
problem in current biology, the solution to which holds great
therapeutic
promise. Fluorescence-Activated Cell Sorting (FACS), a high-throughput
single-cell gene expression assay, has for decades been the key
enabling
technology spurring progress towards this goal. By mapping cell
population
structure in gene expression space, FACS has made it possible to
outline the
branched sequence of fate decisions leading from the hematopoietic stem
cell to
the specialized effector cells of the blood. Because FACS measures
surface-protein expression, it does not provide direct insight into the
decision-making logic driving cellular differentiation, which is
mediated by
transcription factors acting in the nucleus. With today's technology,
the status
of this gene regulatory network is most readily characterized at the
RNA rather
than the protein level. Quantification of mRNA in individual cells
remains
challenging, especially if we seek to measure low-abundance messages
(such as
those for transcriptional regulators), assay multiple targets in the
same
sample, and collect data on enough cells to resolve distinct
sub-populations. A
new technique, microfluidic digital RT-PCR, facilitates quantitation of
rare
transcripts in single cells, and is compatible with low-order
multiplexing
using TaqMan chemistry. Two-stage qRT-PCR is another, less costly
approach
which relies on standard qPCR technology. In this method, a multiplexed
RT-PCR
with a limited number of amplification cycles is followed by
independent
simplex qPCR analyses on aliquots of ‘pre-amplified’ first-round
product. The
protocol is labor-intensive, however, limiting throughput. It will soon
be
feasible to combine two-stage qRT-PCR with microfluidic chip technology
to
support large-scale gene expression surveys, enabling multi-parametric,
cross-sectional analysis of regulatory network state in progenitor cell
populations. This should significantly benefit developmental studies. LightCycler®
480 Real-Time PCR System: Innovative
Solutions for High Throughput PCR. O. Geulen, G. Tellmann Roche
Diagnostics, Roche Applied Email:
Oliver.Geulen@Roche.com The LightCycler® Real-Time PCR Systems from Roche Applied Science have set standards for maximum flexibility, high speed and outstanding data accuracy. The latest innovation, the LightCycler® 480 Real-Time PCR System, continues this tradition and extends it to higher throughputs (96-well / 384-well format) by using plate-based analysis format. The modern instrument design, outstanding technical and software features, as well as advanced reagents and disposables of the LightCycler® System pave the way for high-performance real-time PCR analysis. The LightCycler® 480 software provides versatile solutions for the most common real-time PCR applications like gene detection, gene expression and genotyping analysis. The system’s basic software package comprises basic software and application-specific modules which provide diverse methods for innovative analysis workflows. Furthermore, the system now offers a new analysis tool for high-resolution melting curve analysis (HRM), providing an innovative method to scan genes for unknown variations. A
GPCR brain map using Taqman Low Density Arrays. Samaha R. Applied Email:
samaharr@appliedbiosystems.com The G
Protein-Coupled Receptors
(GPCRs) are a broad class of transmembrane proteins. A large percentage
of
successful small molecule drugs specifically target proteins in this
class. Our
study is designed to generate an expression map of 368 GPCR genes in
the human
brain. We used the Applied Biosystems Human GPCR Panel Taqman® Low
Density
Array (TLDA) and RNA samples from whole brain and 35 different brain
sub-regions to generate this map. A total of 72 TLDA cards, 27,648
individual
qRT-PCR reactions, were run in this study. To make it possible to run
this
large number of qRT-PCR assays from limited starting samples, we used a
multiplex preamplification strategy (Taqman® PreAmp Master Mix
& TLDA
matched oligos). We present a performance analysis for the GPCR TLDA
and preamplification
methodology as well as the GPCR brain map. Several genes show
interesting
tissue distributions constituting potentially interesting drug targets. BioMark™
System: A Breakthrough Real-time qPCR System
for HT Expression Profiling, MicroRNA Analysis, and Single-Cell qPCR. Unger M. Fluidigm
Corp., Email:
marc.unger@fluidigm.com The BioMark
System miniaturizes and
highly parallelizes qPCR in nanofluidic circuits called dynamic arrays
and
digital arrays. Dynamic arrays enable parallel multiplexing of up to 48
genes
for each of 48 samples yielding 2,304 ten nanoliter qPCR reactions per
run. The
dynamic array configuration is completely flexible allowing scientists
to input
any set of 48 samples and any set of 48 genes. The efficiencies
realized in
terms of time, effort, and cost are revolutionary compared to
traditional
microplate based systems. Digital arrays are a novel tool for easily
and
reliably performing digital PCR: single target molecule PCR by limiting
dilutions. Digital arrays provide single-copy sensitivity combined with
extreme
accuracy to enable new applications such as single-cell gene expression
analysis and somatic cell mutation detection. Speed
matters – Fast ways from template to result. Thorsten
Traeger QIAGEN GmbH, Email:
thorsten.traeger@qiagen.com Protocols for
fast PCR provide an
effective means of increasing assay throughput and significantly
reducing the
time required to go from nucleic acid template to final result.
However, fast
PCR using standard PCR chemistries has until now suffered from reduced
sensitivity as well as increased variability. This talk will focus on
how to
overcome the challenges of achieving fast PCR, both in real-time PCR
and RT-PCR
analyses, using a novel technology that is fully compatible with
existing PCR
assays and allows fast PCR on standard as well as fast PCR platforms.
Furthermore, an alternative approach to speed up the experimental
workflow when
analyzing cultured cells will be presented. This approach eliminates
the need
for RNA purification and allows real-time RT-PCR analysis direct from
cell
lysates. The
presentation will expound the
principles of the technologies and will show data demonstrating
successful
real-time and end-point PCR analyses with time savings of up to 70%.
The
real-time PCR data will include data generated using different
detection
formats, such as SYBR Green dye and sequence-specific probes in single
and
multiplex PCR. Jay W. Shin1, Kentaro
Kajiya1, Weiniu Gan2, Peter Li2,
Rainer
Kunstfeld3 and Michael Detmar1 1Institute of
Pharmaceutical
Sciences, ETH Email:
jay.shin@pharma.ethz.ch Angiogenesis
and lymphangiogenesis
have important roles in cancer progression and inflammatory diseases,
but it
has been a challenge to evaluate these processes quantitatively. Using
two
different microarray platforms, we first identified the comprehensive
lineage-specific transcriptomes of human lymphatic (LEC) and blood
vascular
(BVEC) endothelial cells. We identified 236 lymphatic signature genes
and 342
signature genes for blood vascular endothelium. In silico analyses of
the
biologic pathways associated with these genes revealed lineage-specific
functions for each cell type. Using a selection of 85 identified
lineage-specific genes, we developed a TaqMan RT-PCR-based,
microfluidic
card-formatted low-density microvascular differentiation array (LD-MDA)
that
can be used to reliably identify and quantify endothelial cells based
on their
lineage-specific differentiation. Importantly, using LD-MDA, we were
able to
quantify levels of angiogenesis and lymphangiogenesis in tissue samples
from
patients with chronic inflammatory skin disease, indicating that this
assay
might be developed as a novel tool for the rapid and quantitative
evaluation of
pathological (lymph)angiogenesis in clinical and pre-clinical studies. GeXP
- a new approach in high-throughput gene
expression analysis. Han-Chang
Chi1, Yong Wu1, Jane Luo1,
Kahuku Oades2, Gordon Vansant2, Scott K. Boyer1, Manfred Souquet1
and
Keith Roby1, 1Nucleic Acid Testing Business Group, Beckman Coulter,
Inc., 4300
N Harbor Blvd, Fullerton, CA 92834 and 2Analytical Services, Althea Tech Email:
msouquet@beckman.com Quantitative
gene expression
analysis is playing an increasingly important role in cancer research.
Currently available techniques either utilize microarray to detect the
expression
of a high number of genes per reaction at high cost, or utilize
real-time PCR†
to detect the expression of a few genes at low throughput. A multiplex
approach
to analyze a set of multiple genes from a given biological pathway in a
single
reaction using a limited amount of total tissue RNA is of great
interest to
cancer biologists. To address this need, the GenomeLab™ GeXP Genetic
Analysis
System was developed utilizing eXpress Profiling, a patented, highly
multiplexed PCR approach to quickly and efficiently look at the
expression of
20-30 multiplexed gene sets with greater sensitivity. The throughput is
scalable from 80 to over 4000 gene expression results per day. Here we
present
a study of two cancer panels: a set of 29 genes directly or indirectly
related
to breast cancer formation; and the second set of 20 genes for
metastasis
progression. These two multiplex gene expression marker panels were
developed
not only for breast cancer detection, but also to differentiate
cancerous
stages. Our study demonstrated that this novel approach can
quantitatively
detect the expression of both cancer marker panels in a single reaction
using
as little as 25 ng of total RNA isolated from human breast cancer
tissues and
other types of cancerous tissues. The GenomeLab GeXP System has proven
to be a
cost-effective way of performing multiplex gene expression analysis
with
scalable throughput capacity, high assay robustness, and excellent data
quality. † The PCR
process is covered by
patents owned by Roche Molecular Systems, Inc. and F. Hoffman-LaRoche,
Ltd. Novel
qPCR methods for cellular high throughput
compound screenings of SOST expression inhibitors. Angela
Furrer, Simone Degen, Heidi
Jeker, Johann Wirsching and Hansjörg Keller Bone &
Cartilage Unit,
Musculoskeletal Disease Area, Novartis Institutes for BioMedical
Research, Email:
hansjoerg.keller@novartis.com The SOST gene
encodes for
sclerostin, an osteocyte-secreted important negative regulator of bone
mass.
Lack of SOST expression in bone causes the rare human genetic disorders
Sclerosteosis and Van Buchem disease, which are characterized by
massive
life-long bone overgrowth of the whole skeleton. Thus, inhibiting
sclerostin
expression represents a new opportunity for the development of strongly
needed
novel bone anabolic osteoporosis therapies. To this end, we have
established a
cellular assay for the compound screening of SOST expression inhibitors
using
SOST-expressing UMR-106 rat osteosarcoma cells and quantitative
real-time
RT-PCR (qPCR) analysis of SOST mRNA expression. Forskolin inhibited
SOST
expression within 6h by about 90% with an IC50 of 515
± 121 nM. QPCR
analysis was performed using purified RNA and two-step cDNA
synthesis/real-time
PCR, and, thus, did not permit high throughput application. Here, we
report the
testing of two novel methods for faster and simpler qPCR: The FastLane
Cell
Multiplex kit from Qiagen allowing one-step RT-PCR directly from cell
lysates
and another kit from Qiagen permitting fast RT-PCR using thermal
cyclers with
standard or rapid ramping rates. Method performances were investigated
by
determining SOST inhibition dose-response curves for forskolin and
solvent
control. Compared to the two-step qPCR assay with purified RNA, the
one-step
qPCR assay with cell lysates showed very similar data reproducibility
and
accuracy, but was much less laborious requiring less than half the
amount of
time. Solvent control data variation and forskolin IC50
values were
not significantly different between the two methods. Cell lysates were
stable
at -80º C for at least one month allowing qPCR re-analyses or
expression
analyses of new genes. A test kit version for fast RT-PCR was also
successfully
tested yielding similar data reproducibility and variation. Solvent
control
data variation and forskolin IC50 values were not
significantly
different from the previous two methods. RT-PCR time requirement was
further
reduced with this method due the reduced cycling times, but this method
still
required RNA purification. In conclusion, novel qPCR methods were
established
that allow gene expression analysis using cell lysates without prior
RNA
isolation and that enable fast-cycling RT-PCR using normal thermal
cyclers,
respectively. These methods decisively accelerate and simplify cellular
qPCR assays
permitting facilitated automation and thus, they are well suited for
use in
high throughput compound screenings of gene expression regulators. A
comprehensive and quantitative way : HiCEP. Abe M. National
Institute of Radiological
Sciences, Email:
abemasum@nirs.go.jp qPCR is an ideal way to observe the expression of each transcript but is not suitable for comprehensive study, because of the limitation on its throughput. We have attempted to develop a new way to satisfy both requirements, namely highly quantitative and comprehensive. A PCR-based method called AFLP was developed for analyzing numerous DNA fragments formed by restriction-enzyme digestion. Its core technology is a unique PCR step for dividing the whole DNA-fragments population into subpopulations through selective amplification. It is called as ‘selective PCR’ but it had worked still partially. We found that there is temperature point, at which ‘selective PCR’ works nearly perfect. Around 71.5 ºC is good for the annealing temperature where only few miss annealing occur. Cloning and sequencing of more than 15,000 peaks detected in mouse embryonic stem (ES) cells revealed that 96.2% of them were generated with correct selection. In combination with using restriction enzymes, this nearly-100% accurate selective PCR enables us to distinguish huge DNA fragments without any sequence information. This procedure is applicable to transcriptome analysis, genome analysis such as restriction-enzyme mapping and comprehensive DNA methylation assay and so on. Using this procedure, more than 40,000 transcripts including about 2,000 of novel transcripts, were identified in mouse ES cells and the expression of each transcript was measured quantitatively. It is notable that this new procedure can identify unknown transcripts as well as known ones. Non-coding transcripts are often detected. Currently, 100 cells are enough for the analysis. Session: qPCR NOS Session
Normalization
& Optimization & Standardization Chair: T. Nolan /
P. Pinzani Lecture
hall HS 15 Quantification
of mRNA using the real-time RT-PCR. Tania Nolan1, Rebecca E
Hands2, Stephen A Bustin2 1Sigma Email:
tnolan1@europe.sial.com The real-time
reverse transcription
(RT) polymerase chain reaction (PCR) (RT-qPCR) addresses the evident
requirement for quantitative data analysis in molecular medicine,
biotechnology, microbiology, diagnostics and other areas and has become
the
method of choice for the quantification of mRNA. Although often
described as a
“gold” standard, it is far from being a routine assay. Current
problems The
widespread use of this
technology has resulted in the development of numerous protocols that
generate
quantitative data using • fresh,
frozen or archival
(formalin-fixed, paraffin-embedded) samples • whole
tissue biopsies,
microdissected samples, single cells, tissue culture cells • total or
mRNA • a range of
different cDNA priming
strategies • different
enzymes or enzyme
combinations • assays of
variable efficiency,
sensitivity and robustness • diverse
detection chemistries,
reaction conditions, thermal cyclers and • individual
analysis and reporting
methods. This obvious
lack of standardisation
at every step of the assay is exacerbated by significant differences in
sample
processing, use of controls, normalisation methods and quality control
management and has serious implications for the reliability of the
assay,
relevance and reproducibility. The
significant problems caused by
variability of RNA template, assay design and protocols as well as
inappropriate data normalisation and inconsistent data analysis are
widely
known, but also widely disregarded. As a first step towards
standardisation, a
comprehensive RT-qPCR protocol is described that illustrates the
essential
technical steps required to generate quantitative data that are
reliable and
reproducible. qPCR
pitfalls - primer and probe design / fluorophore
quencher combinations. Beslin C. Email:
cl.beslin@eurogentec.com When
designing a qPCR assay,
different parameters have to be taken into account such as
samples/experimental
group, type of controls, design and synthesis of the primer and probe,
method
of RNA extraction (quality and purity of RNA), reverse transcription
(one step
or two step) and qPCR (singleplex or multiplex). All these steps are
commonly
discussed except the design of probes and primers. Primer and
probe design is a crucial
step in your experimental design; time spent in the design of the
primers and
probes will save time in the optimisation of the assay. Design, not
only
includes the basic rules, pitfalls or tricks to design primers and
probes but
also a combination of the right fluorophores and quenchers: which
fluorophore
should be chosen, which quencher should be combined, how to multiplex
them.
These choices are not just dependant on your assay and your instrument
but also
on the available synthesis techniques. Design pitfalls and tricks for
various
instruments and fluorophore-quencher combinations will be discussed. The
Intricacies of Multiplexing Revealed through a
Pathogen Detection Assay. V. Evan
Messenger, Ben Sowers Biosearch
Technologies, Email:
evan@biosearchtech.com The
intensifying demand to detect
pathogens in food products, agriculture, and the environment can be met
with
speed and confidence by multiplexing PCR assays together.
Spectrally-distinct
fluorophores and quenchers provide the ability to detect multiple
genetic
signatures that distinguish closely related strains, all within the
same
reaction chamber. Here we present amplifications from a TaqMan®
assay
engineered to detect the virulence factors of Bacillus anthracis, and
identify
several common laboratory strains. Using this assay as an example, we
provide a
step-by-step tutorial on the considerations that go into the design and
optimization of multiplexed PCR: selecting sequences, optimizing
reactions, and
characterizing amplification performance. By sensitively exposing the
presence
of these organisms, we highlight the capability of multiplexed qPCR. Olbrich M. GSF, Email:
maren.olbrich@gsf.de Analyses of
different plant
stressors are often based on gene expression studies. Quantitative
Real-time
RT-PCR (qRT-PCR) is the most sensitive method for the detection of low
abundance genes. However, a critical point to note is the selection of
housekeeping genes as an internal control. Many so-called ‘housekeeping
genes’
are often affected by different stress factors and may not be suitable
for use
as an internal reference. We tested seven housekeeping genes of
European beech
by qRT-PCR using the Sybr Green PCR kit. Specific primers were designed
for 18S
rRNA, actin, GAPDH1, GAPDH2, α-tubulin, and ubiquitin-like protein.
Beech saplings
were treated with increased concentrations of either ozone or CO2. In
parallel,
the expression of these genes was analyzed upon pathogen infection with
Phytophthora citricola. To test the applicability of these genes as
internal
controls under realistic outdoor conditions, sun and shadow leaves of
60-year-old trees used as a comparison. The regulation of all genes was
tested
using a linear mixed-effect model of the R-system. Results from
independent
experiments showed that actin was the only gene not affected by any
treatment
tested. The expression of the other housekeeping genes varied more or
less with
the degree of stress applied. These results highlight the importance of
undergoing an individual selection of internal control genes for
different experimental
conditions. EvaGreen:
a new fluorescent nucleic acid dye for
real-time qPCR. Mao F1,2,
Leung WY1,
and Xin X.1,2 1Biotium,
Inc., Email:
shanex@allelogic.com EvaGreen (EG)
is a newly developed
DNA-binding dye that is particular useful for both quantitative
real-time PCR
(qPCR) and post PCR DNA melt curve analysis. Compared with SYBR Green I
(SG),
EG was found to have less PCR inhibition and thus could be used at a
relatively
high concentration (0.67-1.33 uM), which enabled PCR to yield more
robust
signals without adversely affecting the specificity and amplification
efficiency. The use of relatively high EG concentration also eliminated
so-called dye redistribution problem, making the dye suitable for use
in
high-resolution melt curve analysis. Furthermore, the relatively low
PCR
inhibition of EG also permitted fast cycling protocol in qPCR without
sacrificing PCR specificity and reproducibility. Because EG has
excitation and
emission spectra similar to those of SG and FAM, the dye is compatible
with
nearly all commercial qPCR instruments. Finally, EG was shown to be
nonmutagenic, cell membrane-impermeable and extremely stable, thus
facilitating
its use and handling. Quantitative,
multiplexed amplification with the
Plexor™ qPCR Systems. Katharine
Hoffmann, Benjamin Krenke,
Cynthia Sprecher, Susan Frackman, Ethan Strauss, and Douglas Storts Promega
Corporation, Email:
doug.storts@promega.com The Plexor™
qPCR, One-Step qRT-PCR,
and Two-Step qRT-PCR Systems enable quantitative, multiplex
amplification
experiments on a variety of real-time instruments. The system consists
of free
web-accessible primer design software, reagent systems and free data
analysis
software. The Plexor™ Systems work by measuring a reduction in
fluorescent
signal during amplification. Amplification uses only two primers, one
of which
contains a modified base with a fluorescent reporter on the 5'-end. As
amplification proceeds, fluorescence is reduced by site-specific
incorporation
of a fluorescent quencher (attached to a modified nucleotide) inserted
opposite
the complementary modified base. The quencher is in close proximity to
the
fluorescent dye located on the 5'-end of the primer, resulting in a
reduction
in the fluorescent signal. After PCR, a melt analysis can be performed
to
provide an internal control for the final assay design or to expedite
troubleshooting during assay development. We will present data
demonstrating
reliable multiplexed amplification results, over a broad range of
target
concentrations. R. Powell1, T.Brown2,
J. Wicks1 1PrimerDesign
Ltd, A novel probe (PerfectProbeTm) has been designed which incorporates the best functional characteristics of Taqman and molecular beacons (MB) probes. These probes have a “shared stem” hair pin structure and are therefore optimally quenched like MB probes. However they are cleaved, rather than displace, during amplification like the Taqman probe. These characteristics combine to produce a probe that is more sensitive at detecting amplification than either Taqman or Molecular Beacon probes. The novel probe produces earlier cycle threshold values when compared directly to the reporting of Taqman or MB probes. They also give rise to much higher signal to noise ratios and are more flurogenic. The critical design criteria for these probes will be presented and a model for understanding their functioning during real-time PCR amplification. Comparison
of different probe chemistries and
platforms to improve the sensitivity of real-time PCR. Reynisson E.1,2, Josefsen
M.H.3,
Krause M.3, Hoorfar J.3 1Matís,
Email:
eyjolfur@matis.is A validated
qPCR-based Salmonella method targeting a 94-bp sequence of the
ttr gene was used as
a model to
compare six different combinations of reporter and quencher dyes of a
TaqMan
probe, on three different instruments, to improve the detection limit
in a
realtime PCR assay with the aim of a same-day analysis. The use of
locked nucleic
acids (LNA) and Scorpion probes were also tested. The combination
FAM–BHQ1 or
Cy5–BHQ3, both dark quenchers, gave the best results (Cycle threshold
(Ct) of
25.42 ± 0.65 and 24.47 ± 0.18 at 103 DNA
copies). When comparing
different probe technologies, the LNA probe (FAM–BHQ1) was the most
sensitive
with the strongest fluorescence signal (dR last 48066), resulting in
0.6 to 1.1
lower Ct values than TaqMan probe, and 1.9 to 4.0 lower Ct than the
Scorpion
system (FAM–BHQ1). The RotorGene real-time PCR instrument gave 0.4–1.0
lower Ct
values (more sensitive) than the Mx3005p, and 1.5–3.0 lower than the
ABI 7700.
Using the LNA in a RotorGene instrument, we detected the following
Salmonella
DNA copies in 1-ml pre-enriched samples: fishmeal (100 copies), chicken
rinse
(100 copies) and pig feces (10 copies). The detection probability of
the final
assay on inoculated fecal samples was 100% at 2 x 104 copies
per ml.
In conclusion, the LNA probe with annealing temperature of 65 °C
could be
useful for more sensitive detection limits. Sample
number and denaturation time are crucial for
the accuracy of capillary-based LightCyclers. Thomas von
Kanel, Florentin
Adolf, Mircea Schneider, Javier Sanz, Sabina Gallati Division of
Human Genetics, Email:
biotom@students.unibe.ch Background: While
performing real-time PCR for deletion and
duplication screenings on a capillary-based LightCycler instrument,
carousel-position variations in cycles over threshold Ct
were
observed. These variations can reach an extent indicating a duplication
or even
a deletion in a diagnostic setup (maximum Ct difference
between
different carousel positions > 0.5). Methods: Protocol
amendments such as complete filling of the
carousel with capillaries and increased denaturation time during PCR
cycling
were tested for their potential to increase the accuracy of LightCycler
instruments. Results: Highest
accuracy of the LightCycler instruments was
obtained by complete filling of the carousel and by increasing the
denaturation
time to 5 seconds during cycling, with coefficients of variation
dropping from
≥ 0.68% (unfilled carousel, 0 seconds denaturation) to ≤ 0.23% (filled
carousel, 5 seconds denaturation), thus minimizing the chance to obtain
false-positive results due to positional effects. Any
changes ??? => please contact the scientific
organizer Michael W. Pfaffl via qPCR2007@wzw.tum.de
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