|
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
|