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Accuracy and
calibration of commercial oligonucleotide
and custom cDNA microarrays
Yuen
T, Wurmbach E, Pfeffer RL, Ebersole BJ, Sealfon SC.
Department of Neurology, Mount Sinai School of Medicine, New
York, NY 10029, USA.
Nucleic
Acids Res. 2002 30(10): e48.
We
compared the accuracy of
microarray measurements
obtained with oligonucleotide arrays (GeneChip, Affymetrix) with a
laboratory-developed cDNA array by assaying test RNA samples from an
experiment using a
paradigm known to regulate many genes measured on both arrays. We
selected
47 genes represented on both arrays, including both known regulated
and unregulated transcripts, and established reference relative
expression
measurements for these genes in the test RNA samples using quantitative
reverse transcriptase real-time PCR (QRTPCR) assays. The validity of
the reproducible (average coefficient of variation = 11.8%) QRTPCR
measurements
were established through application of a new mathematical model. The
performance
of both array platforms in identifying regulated and non-regulated
genes
was identical. With either platform, 16 of 17 definitely regulated
genes
were correctly identified, and no definitely unregulated transcript was
falsely identified as regulated. Accuracy of the fold-change
measurements
obtained with each platform was assessed by determining measurement
bias.
Both platforms consistently underestimate the relative changes in mRNA
expression between experimental and control samples. The bias observed
with cDNA arrays was predictable for fold-changes <250-fold by
QRTPCR
and could be corrected by the calibration function F(c) =
F(a(cDNA))(q),
where F(a(cDNA)) is the microarray-determined fold-change comparing
experimental
with control samples, q is the correction factor and F(c) is the
calibrated
value. The bias observed with the commercial oligonucleotide arrays was
less predictable and calibration was unfeasible. Following calibration,
fold-change measurements generated by custom cDNA arrays were more
accurate than those obtained by commercial oligonucleotide arrays. Our
study demonstrates systematic bias of microarray measurements and
identifies a calibration
function that improves the accuracy of cDNA array data.
Focused
microarray analysis.
Wurmbach E, Yuen T, Sealfon SC.
Methods. 2003 31(4): 306-316.
Department of Neurology, Mount Sinai School of Medicine, Box
1137, 1 Gustave L. Levy Place, New York, NY 10029, USA.
We
describe detailed protocols and
results with an integrated platform
for studying relative transcript expression, including microarray
design and fabrication, analysis and calibration algorithms, and high
throughput quantitative real-time PCR. This approach optimizes
sensitivity and accuracy while controlling the cost of experiments. A
high quality cDNA array was fabricated using a restricted number of
carefully selected transcripts with each clone printed in triplicate.
This focused array facilitated both repeated measurement and replicate
experiments. Following normalization and differential expression
analysis, we found that experiments with this array identified
differentially expressed transcripts with a high degree of accuracy and
with high sensitivity to low levels of differential expression. Using a
calibration algorithm improved the accuracy of the array in quantifying
the relative level of transcript expression. All differentially
expressed transcripts identified by the array were independently tested
using high
throughput quantitative real-time PCR assays. This approach reliably
identified
transcripts having as low as 1.3-fold differences in transcript
expression
between RNA samples from treatment- and control groups and was
applicable
to highly heterogenous tissue sources such as hypothalamus and cerebral
cortex.
Real-time RT-PCR
profiling of over 1400 Arabidopsis transcription factors:
unprecedented
sensitivity reveals novel root- and shoot-specific genes.
Czechowski T, Bari RP, Stitt M, Scheible WR, Udvardi MK.
Plant J. 2004 Apr;38(2):366-79.
Max-Planck Institute of Molecular Plant Physiology, Am Muhlenberg 1,
14476 Golm, Germany
Summary
To overcome the detection
limits inherent to DNA array-based methods of transcriptome analysis,
we
developed a real-time reverse transcription (RT)-PCR-based resource for
quantitative measurement of transcripts for 1465 Arabidopsis
transcription factors
(TFs). Using closely spaced gene-specific primer pairs and SYBR Green
to
monitor amplification of double-stranded DNA (dsDNA), transcript levels
of 83%
of all target genes could be measured in roots or shoots of young
Arabidopsis
wild-type plants. Only 4% of reactions produced non-specific PCR
products. The
amplification efficiency of each PCR was determined from the log slope
of
SYBR Green fluorescence versus cycle number in the exponential phase,
and was
used to correct the readout for each primer pair and run. Measurements
of
transcript abundance were quantitative over six orders
of magnitude, with a
detection limit equivalent to one transcript molecule in 1000 cells.
Transcript levels for
different TF genes ranged between 0.001 and 100 copies per cell. Only
13% of
TF transcripts were undetectable in these organs. For comparison, 22K
Arabidopsis Affymetrix chips detected less than 55% of TF transcripts
in the same
samples, the range of transcript levels was compressed by a factor more
than
100, and the data were less accurate especially in the lower part of
the response
range. Real-time RT-PCR revealed 35 root-specific and 52
shoot-specific TF genes, most of which have not been identified as
organ-specific
previously. Finally, many of the TF transcripts detected by RT-PCR are
not
represented in Arabidopsis EST (expressed sequence tag) or Massively
Parallel
Signature Sequencing (MPSS) databases. These genes can now be annotated
as expressed.
Gonadotropin-releasing
hormone receptor-coupled gene
network
organization.
Wurmbach E, Yuen T, Ebersole BJ, Sealfon SC.
Department of Neurology, Mount Sinai School of Medicine, New
York, NY 10029, USA
J Biol Chem. 2001 ;276(50): 47195-47201
An
early gene cDNA microarray was
developed to study genes that are
regulated immediately following gonadotropin-releasing hormone (GnRH)
receptor activation. 956 selected candidate genes were printed in
triplicate, a t statistic-based regulation algorithm was used for data
analysis,
and the response to GnRH in a time course from 1 to 6 h was determined.
Measurements were highly reproducible within arrays, between arrays,
and between experiments. Accuracy and algorithm reliability were
established
by real-time polymerase chain reaction assays of 60 genes. Gene changes
ranging from 1.3- to 31-fold on the microarray were confirmed by
real-time
polymerase chain reaction. Many of the genes were found to be highly
regulated.
The regulated genes identified were all elevated at 1 h of treatment
and
returned nearly or completely to baseline levels of expression by 3 h
of
treatment. This broad, robust, and transient transcriptional response
to
constant GnRH exposure includes modulators of signal transduction (e.g.
Rgs2 and IkappaB), cytoskeletal proteins (e.g. gamma-actin), and
transcription
factors (e.g. c-Fos, Egr1, and LRG21). The interplay of the activators,
repressors, and feedback inhibitors identified embodies a combinatorial
code to direct the activity of specific downstream secondary genes.
Effect of zinc deficiency on the mRNA
expression pattern in liver and jejunum of adult rats:
Monitoring
gene expression using cDNA microarrays combined with
real-time RT-PCR
Michael W.
Pfaffl, B. Gerstmayer, A. Bosio, Wilhelm Windisch
Journal of Nutritional Biochemistry 14 (2003) 691–702
Institute
of
Physiology, Department of Animal Sciences,Centre of Life and Food
Sciences,
TUM, 85354 Freising, Germany
Memorec Biotech GmbH, Medical Molecular Research Cologne, 50829, Koeln,
Germany
Division of Animal Nutrition and Production Physiology, Department of
Animal Sciences, Centre of Life and Food Sciences, TUM, 85354,
Freising, Germany
In the
study
presented here, the effect of zinc deficiency on mRNA expression levels
in
liver and jejunum of adult rats was analyzed. Feed intake was
restricted to 8 g/day. The semi-synthetic diet was fortified with pure
phytate
and contained either 2 g Zn/g (Zn deficiency, n 6) or 58 g Zn/g
(control, n 7). After 29 days of Zn depletion feeding, entire jejunum
and liver were retrieved and total RNA was extracted. Tissue specific
expression pattern were screened and quantified by microarray analysis
and verified individually via real-time RT-PCR. A relative
quantification was performed with the newly developed Relative
Expression Software Tool © on numerous candidate genes which
showed a differential expression. This study provides the first
comparative view of gene
expression regulation and fully quantitative expression analysis
of 35 candidate genes in a non-growing Zn deficient adult rat model.
The expression
results indicate the existence of individual expression pattern in
liver and jejunum and their tissue specific regulation under
Zn deficiency. In addition, in jejunum a number of B-cell related
genes could be demonstrated to be suppressed at Zn deficiency. In
liver,
metallothionein subtype 1 and 2 (MT-1 and MT-2) genes could be shown to
be dramatically repressed and therefore represent putative markers for
Zn
deficiency. Expression results imply that some genes are expressed
constitutively, whereas others are highly regulated in
tissues responsible for Zn homeostasis.
Figure
1: Representative example of a gene expression pattern captured as
an image of a cDNA-array hybridised with Cy3-labelled control sample
(green fluorescence) and Cy5-labelled sample (Zn deficiency in red
fluorescence). Each of the 1001 cDNAs is spotted either
in quadrant A and B. Four replicates for each cDNAs are spotted,
resulting in four A and B quadrants, respectively. A magnification for
the most up-regulated (MT-1 and MT-2) and down-regulated (IL-6R-beta)
gene transcripts is shown.
Figure
2: Frequency and level of down- or up-regulation of regulated
genes of microarray experiments in liver and jejunum of Zn deficiency
rats. Frequency plot of both tissue expression pattern exhibit a three
parametric Gaussian distribution (p <0.0001). Mean (µ) and
boarders of confidential interval are indicated (µ ± 1.96
times the standard deviation of the Gaussian distribution).
Significantly different expressed genes (p<0.05) were selected
outside the 95% confidential interval. Lines indicate an approximation
of
95% interval in liver and jejunum.
Identification of
Genes Responsive to Intracellular Zinc Depletion in the Human
Colon
Adenocarcinoma Cell Line HT-29.
Kindermann B, Doring F, Pfaffl M, Daniel H.
J Nutr. 2004 Jan;134(1): 57-62.
Molecular Nutrition Unit and. Department of Animal Sciences, Technical
University of Munich, D-85350 Freising-Weihenstephan, Germany.
Zinc
is essential for the
structural and functional integrity of cells and plays a pivotal role
in the control of
gene expression. To identify genes with altered mRNA expression level
after zinc
depletion, we employed oligonucleotide arrays with approximately 10,000
targets
and used the human colon adenocarcinoma epithelial cell line HT-29 as a
model. A low intracellular zinc concentration caused alterations in the
steady-state mRNA levels of 309 genes at a threshold factor of 2.0.
Northern blot
analysis and/or real-time RT-PCR confirmed the array results for 12 of
14
selected targets. Genes identified as regulated based on microarray
data encode mainly
proteins involved in central pathways of intermediary metabolism (79
genes)
including protein metabolism (21). We also identified five groups of
genes
important for basic cellular functions such as signaling (30), cell
cycle control
and growth (15), vesicular trafficking (15), cell-cell interaction
(13),
cytoskeleton (10) and transcription control (19). The latter group
comprises several
zinc finger-containing transcription factors of which the Kruppel-like
factor 4
showed the most pronounced changes. Western blot analysis confirmed the
increased expression level of this protein in cells grown under low
zinc conditions.
Our findings in a homogenous cell population demonstrate that the
molecular
mechanisms by which cellular functions are altered at a low zinc
status,
occur via pleiotropic effects on gene expression. In conclusion, the
pattern of
zinc-affected genes may represent a reference for further studies to
define the zinc
regulon in mammalian cells.
Validation
of array-based gene expression profiles by
real-time (kinetic) RT-PCR.
Rajeevan
MS, Vernon SD, Taysavang N, Unger
ER. (2001)
J Mol Diagn. 2001
Feb;3(1):26-31.
We
evaluated real-time (kinetic) reverse
transcription-polymerase chain reaction (RT-PCR)
to validate differentially expressed genes identified by DNA arrays. Gene
expression of two keratinocyte subclones differing in the physical
state of human
papillomavirus (episomal or
integrated) was used as a model system. High-density
filter arrays identified 444 of 588 genes as either negative
or expressed
with less than twofold difference, and the other 144 genes as
expressed
uniquely or with more than twofold difference between the two
subclones.
Real-time RT-PCR used LightCycler-based SYBR Green I dye detection
and
melting curve analysis to validate the relative change in gene
expression. Real-time
RT-PCR confirmed the change
in expression of 17 of
24 (71%) genes identified
by high-density filter arrays. Genes with strong hybridization
signals
and at least twofold difference were likely to be validated by real-time
RT-PCR.
This data suggests that (i) both hybridization intensity and the level
of
differential expression determine the likelihood of validating
high-density filter
array results and (ii) genes
identified by DNA arrays with a two- to fourfold
difference in expression cannot be eliminated as false nor be accepted
as
true without validation. Real-time RT-PCR based on LightCycler
technology is well-suited
to validate DNA array
results because it is quantitative, rapid, and requires
1000-fold less RNA than conventional assays.
Global
cDNA amplification combined with real-time
RT-PCR:
accurate
quantification of multiple human potassium channel genes at
the single cell level.
Al-Taher A,
Bashein A, Nolan T, Hollingsworth
M, Brady G. (2000)
Yeast. 2000 Sep
30;17(3):201-210.
<>
We have developed a sensitive quantitative RT-PCR procedure
suitable for the analysis
of small samples, including
single cells, and have used it to measure levels of
potassium channel mRNAs in a panel of human tissues and small numbers
of
cells grown in culture. The method involves an initial global
amplification of
cDNA derived from all added
polyadenylated mRNA followed by quantitative RT-PCR of
individual genes using specific primers. In order to facilitate rapid
and
accurate processing of samples, we have adapted the approach to allow
use of TaqMan
real-time quantitative PCR. We
demonstrate that the approach represents a major
improvement over existing conventional and real-time quantitative PCR
approaches, since it can be applied to samples equivalent to a single
cell, is able to
accurately measure expression
levels equivalent to less than 1/100th copy/cell
(one specific cDNA molecule present amongst 10(8) total cDNA
molecules).
Furthermore, since the initial step involves
a global amplification of all
expressed genes, a permanent cDNA archive is generated from each sample,
which
can be regenerated indefinitely for further expression
analysis.
DNA
microarrays and beyond: completing the journey from
tissue to cell.
Mills JC,
Roth KA, Cagan RL, Gordon JI.
(2001)
Nat Cell Biol
2001 Aug;3(8):E175-178
Erratum
in: Nat Cell Biol 2001 Oct;3(10):943
For
the cell
biologist, identifying changes in gene expression using DNA microarrays is just
the start of a long journey from tissue to cell. We discuss how chip users can
first filter noise (false-positives) from daunting microarray datasets. Combining
laser capture microdissection with real-time polymerase chain reaction and reverse
transcription is a helpful follow-up step that allows expression of selected genes to
be quantified using sensitive new in situ hybridization
and
immunohistochemical methods based on tyramide signal amplification.
Use
of real-time quantitative PCR to validate the results of
cDNA array
and
differential display PCR technologies.
Rajeevan MS,
Ranamukhaarachchi DG, Vernon SD, Unger
ER. (2001)
Methods. 2001
Dec;25(4):443-51.
Real-time
reverse
transcription polymerase chain reaction (RT-PCR) methods that monitor
product accumulation
were adapted for the validation of differentially expressed genes. We
describe a real-time
quantitative PCR assay that uses SYBR Green I dye-based detection and
product melting curve analysis to validate
differentially expressed genes
identified by gene expression profiling technologies.
Since SYBR Green
I dye is a nonspecific intercalating dye, the reaction is made specific by using
"hot-start" PCR and empirically determined annealing and signal
acquisition temperatures
for each gene-specific primer. Relative expression levels were
quantified by constructing a standard curve
using cDNA dilutions of a
highly expressed gene. Using this
approach, real-time
PCR validated 17 of 21 (71%) genes identified
by DNA arrays, and all but 1 of 13 (91%) genes
identified by
differential display PCR (DD-PCR). Validation of differentially
expressed genes detected by array
analysis was related to hybridization intensity. Real-time RT-PCR
results
suggest that
genes identified by DNA arrays with a two to fourfold
difference in expression cannot be accepted as
true or false
without validation. Validation of differentially expressed genes
detected by DD-PCR
was not affected by band intensities. Regardless of the gene expression
profiling technology (microarrays, DD-PCR,
serial analysis of gene expression and
subtraction hybridization), once the sequence
of gene of interest is
known, the real-time RT-PCR
approach is well suited for validation of differential expression since
it is quantitative and rapid and requires 1000-fold less RNA than
conventional assays.
The
limit fold change model: A practical approach for
selecting
differentially
expressed genes from microarray data.
Mutch DM, Berger A,
Mansourian R, Rytz A, Roberts MA.
BMC
Bioinformatics 2002 Jun 21;3(1):17
Metabolic and
Genomic Regulation, Nestle Research Center,
Vers-chez-les-Blanc,
CH-1000 Lausanne 26,
Switzerland
BACKGROUND:
The
biomedical community is developing new methods of data analysis to more efficiently
process the massive data sets produced by microarray experiments. Systematic and
global mathematical approaches that can be readily applied to a large number of
experimental designs become fundamental to correctly
handle the
otherwise overwhelming data sets.
RESULTS:
The gene selection model presented herein is based on the observation
that:
(1) variance
of gene expression is a function
of absolute expression;
(2) one can
model this relationship in order
to set an appropriate lower fold change limit
of significance; and
(3) this
relationship defines a function that
can be used to select differentially expressed
genes.
The model first
evaluates fold change (FC) across the
entire range of absolute expression levels for
any number of
experimental conditions. Genes are systematically binned, and those genes within the top X% of
highest FCs for each bin are evaluated both with and without the
use of replicates. A
function is fitted through the top X% of each bin, thereby defining a limit
fold change. All genes selected by the 5% FC model lie above measurement
variability using a within standard deviation (SDwithin) confidence
level of 99.9%. Real
time-PCR (RT-PCR) analysis demonstrated 85.7% concordance with microarray data selected
by the limit function.
CONCLUSION:
The
FC model can confidently select differentially expressed genes as corroborated by variance data
and RT-PCR. The simplicity of the overall process permits selecting model limits that
best describe experimental data by extracting information
on gene expression
patterns across the range of expression levels.
Genes selected by this process
can be consistently compared between experiments and
enables the user to
globally extract
information with a high degree of confidence.
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Michael W.
Pfaffl & Rupert M. Bruckmaier (2002)
DNA array and real-time PCR an
optimal combination !
The use of
real-time (kinetic) quantitative PCR to validate cDNA array results
Array
Meeting: COST B20: Mammary development, function and cancer on
10th and 11th of May in Utrecht.
The
application of the real-time (kinetic) PCR to amplify cDNA products
reversely transcribed from mRNA (RT) and microarray technology are on
the way to become routine tools in molecular biology. They are both
well
suited to study gene expression, but each methodology has its specific
advantages and disadvantages. Microarray technology is ideal to screen
a lot of genes in one step (>10,000 gene transcripts) and kinetic
RT-PCR is very sensitive, highly quantitative and requires up to
1000-fold
less RNA. Both allow a relative and accurate quantification of mRNA
molecules with a sufficiently high repeatability and low
variability.
But accurate
quantification of nucleic acids requires
a reproducible methodology and an adequate mathematical model for
data analysis. The particular topics of the relative quantification in
microarray and kinetic PCR technology of a target gene transcript
in comparison to a reference gene transcript or housekeeping gene are
described. Therefore a new mathematical model and software is
presented.
The relative expression ratio ( R ) is calculated from the kinetic PCR
efficiencies (E) and the crossing point (CP) deviation (DCP) of an
unknown
sample versus a control. This model needs no calibration curve. Control
levels were included in the model to standardize each reaction run with
respect to RNA integrity, sample loading and inter-PCR variations. High
accuracy and reproducibility (<2.5% variation) were reached in
LightCycler® RT-PCR using the established mathematical model
(Pfaffl, NAR 2001). R
of a target gene is expressed in a sample versus a control in
comparison
to a reference gene. Etarget = real-time PCR efficiency of target gene
transcript; Eref = real-time PCR efficiency of reference gene
transcript; ?CPtarget = CP deviation of control - sample of target gene
transcript; ?CPref =
CP deviation of control - sample of reference gene transcript.
An Excel®
based Relative Expression Software Tool
(REST ©) is now available, which calculates E and R of various
(<16) samples and four target genes (Pfaffl et al., NAR 2002).
Relative
expression ratio is tested for significance compared to control on the
basis of an Pair Wise Fixed Reallocation Randomization Test ©.
Using this
approach, to screen the tissue specific expression levels by microarray
and confirm the results by kinetic RT-PCR
and REST © is a powerful and optimal combination. The advantages
of both quantification systems were added - high throughput of the
microarray and sensitivity of the real-time RT-PCR.
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Michael W.
Pfaffl, Petros Arnaoutis & Alois Sellmayer
(2002)
Mikroarray und real-time RT-PCR –
eine optimale Kombination ! Verifikation von DNA Array
Ergebnissen
mittels real-time RT-PCR am Beispiel verletzungs-regulierter Gene
in einer HUVEC Zellkultur
Dechema
Statusseminar „Chiptechnologie: Transkriptom – Proteom – Metabolom“,
102-104
Einleitung:
DNA Mikroarrays sind großartige Werkzeuge um funktionelle
Zusammenhänge auf der mRNA Ebene zwischen einer Vielzahl von Genen
zu untersuchen. Aber aufgrund der sequenzabhängigen
Hybridisierungseigenschaften und deren Variationen die den
Hybridisierungsreaktionen zugrunde liegen, müssen die Array
Ergebnisse als semi-quantitativ angesehen werden. Als optimale
Ergänzung der signifikanten Array Ergebnisse bietet sich die
sensitive Verifikation der Expressionsdaten mittels voll quantitativer
real-time RT-PCR an.
Ziel dieses
Projektes bestand darin mittels Northern-Blot, cDNA Array und real-time
RT-PCR Techniken, einen Katalog an Genen zu ermitteln die im Menschen
regional bzw. temporär beschränkte mRNA Expressionsmuster
während der ersten Stunden der Wundheilung aufweisen.
Hintergrund:
Die mechanische Verletzung der Gefäßwand, wie z.B. durch die
Angioplastie, führt zur Aktivierung von Endothelzellen und zum
endothelialen Wundverschluss durch deren Migration und Proliferation.
Eine sehr schnelle Zellantwort auf die mechanische
Endothelzellverletzung stellt die Expression von Early Response Genen,
die eng mit dem Zellwachstum korreliert sind, dar. So induziert die
mechanische Verletzung die Expression von c-FOS mRNA (Briski &
Gillen, 2001) und EGR-1 mRNA (Yan et
al., 2000) und bereits nach einer Stunde ist das Maximum der mRNA
Expression erreicht.
Ziel unserer
Untersuchungen war es in vitro weitere Early Response Gene zu
identifizieren, die durch die mechanische Verletzung in kürzester
Zeit in Endothelzellen aktiviert werden und
dadurch einen besseren Einblick in die molekularen Mechanismen des
Wundheilungsprozesses zu gewinnen. Um dieses Ziel zu erreichen, kamen
unterschiedliche molekularbiologische Methoden zum Einsatz, wie etwa
Northern-Blot (Ergebnisse werden nicht beschrieben), cDNA
Mikroarray Technologie und als voll quantitative
Bestätigungsmethode die real-time RT-PCR.
Methodik:
Um neue durch die mechanische Verletzung induzierte Gene zu entdecken
und zu identifizieren wurde ein Zeitfenster von bis zu 6 Stunden
gewählt. Aus unverletzten und verletzten konfluent gewachsenen
humanen Endothelzellen aus Umbilikalvenen (HUVEC) wurde Gesamt-RNA
extrahiert und mittels des Atlas Pure Total Labeling Sytem (Clontech,
CA, USA) [P32]-markierte cDNA Sonden hergestellt. Diese Sonden wurden
getrennt voneinander auf zwei Atlas Human 1.2 I Array Membranen
(Clontech) hybridisiert: unverletzt versus verletzt cDNA Sonden aus
HUVEC Zellen. Auf diesen Nylon-Membranen sind cDNA Fragmente von 1176
verschiedene Genen immobilisiert, darunter auch Housekeeping Gene zur
Normierung der Expressionsergebnisse, wie GAPDH, b-Actin,
Ubiquitin.
Die
Bestätigung dieser semi-quantitativen Array Ergebnisse erfolgte
mittels voll quantitativer real-time RT-PCR Analyse.
Hierfür wurde mit einem LightCycler (Roche Diagnostics, Penzberg)
gearbeitet, der schnelles Thermocycling (rapid cycling) mit der
Online SYBR Green I Fluoreszenzdetektion der RT-PCR Produkte
kombiniert. Als Quantifizierungsstrategie wurde eine neu entwickelte
normalisierte relative Quantifizierung gewählt (Pfaffl, 2001),
wobei die mRNA
Expressionsdaten eines Zielgens mit denen eines Referenzgens (House
Keeping Gen) verglichen wurden. Die relative Expressionsratio ( R
) wird anhand der PCR Effizienzen (E) und der Differenz der „Crossing
Points“ (CP) der zu vergleichenden Ansätze berechnet. In unserem
Falle waren das einerseits unverletzte versus verletzte Endothelzellen
Gesamt-RNA bzw. cDNA.
Ergebnisse: Die Bestimmung der
CP Zyklenzahlen (n=3) der real-time
RT-PCR für GAPDH und ATF-3 zeigen die geringe intra-Assay
Varianzen (<2,8% VQ) und somit hohe Reproduzierbarkeit der
LightCycler RT-PCR. Für die Zielgene ergaben sich folgenden
minimale und maximale real-time PCR Effizienzen: Ec-FOS = 1,89 und
EICAM-1 = 2,06. Es konnte mit Hilfe der beschriebenen Methoden neben
schon bekannten durch Verletzung induzierte Early Response Gene, wie
c-FOS, und EGR-1, auch weitere Gene nachgewiesen werden, wie ICAM-1,
ATF-3, TTP, ADAMTS-1, ETR-101 und PAI-1 (Arnaoutis et al., 2000). Die
Genexpression wurden im verletzten Zustand, im Vergleich zur
unverletzten Kontrolle, extrem aufreguliert: c-FOS 264–fach, TTP
102-fach, ATF-3 43,5-fach, ADAMTS-1 25,3-fach, ETR-101 11,2-fach,
ICAM-1 5,7-fach und PAI-1 2,2-fach (Mittelwert aus drei
Wiederholungen). GAPDH wurde im Gegensatz nur marginal reguliert
(~40%). Die Expressionsdaten weisen jedoch darauf hin, dass nicht nur
Transkriptionsfaktoren, sondern auch Adhäsionsmoleküle und
Plasminogen Inhibitoren in der Frühphase der mechanischen
Verletzung von Endothelzellen vermehrt expremiert werden.
Schlussfolgerungen:
Mit dem Northern-Blot und dem Atlas DNA Array System konnte die
differentielle Expression von einer Vielzahl von „neuer“ Early Response
Genen aufgezeigt und mittels relativer Quantifizierung in der real-time
RT-PCR exakt quantifiziert werden.
Mit der
Mikroarray Technik und als optimal Ergänzung die real-time RT-PCR
wurden somit schnelle als auch hoch quantitative analytische
Möglichkeiten geschaffen, den Funktionszusammenhang auf mRNA Ebene
zu entdecken, was besonders in nächster Zukunft vor dem
Hintergrund des komplett publizierten humanen Genoms von besonderen
Interesse sein wird.
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Birgit
Kindermann, Hannelore Daniel, Michael W. Pfaffl
& Frank Döring (2002)
Einsatz der cDNA-Array-Technologie
zur Identifizierung Zink-sensitiver Gene in-vitro.
Dechema
Statusseminar „Chiptechnologie: Transkriptom – Proteom – Metabolom“,
S.79
Das
lebensnotwendige Spurenelement Zink wirkt als Cofaktor von mehr als 300
Enzymen und ist ein Stabilisator biologischer Membranen. Zink ist
außerdem Bestandteil von Transkriptionsfaktoren. Es greift somit
in die Regulation der Genexpression ein1. Während in
Modellorganismen wie z. B. Saccharomyces cerevisiae zahlreiche
Zink-sensitive Gene identifiziert wurden2, sind beim Säuger bisher
nur wenige Gene als Zink-abhängig beschrieben worden.
Möglicherweise sind die genutzten Methoden wie z. B. differential
display3 zu unempfindlich, um umfangreiche Daten zur
Zink-abhängigen Expression einzelner Gene zu erhalten. Wir haben
deshalb geprüft, ob die cDNA-Array-Technologie geeignet ist,
Zink-abhängige Säugergene zu identifzieren. Als in-vitro
Modellsystem dienten intestinale Epithelzellen (HT-29), die für 72
Std. in einem Medium mit normaler (0.25 ppm) bzw. erhöhter (10
ppm) Zinkkonzentration kultiviert wurden. Die gewählten
Bedingungen führten zur drastischen Veränderung der
Expression eines bereits bekannten Zink-sensitiven Gens
(Metallothionein-1). Zum Screening dienten cDNA-Arrays (Clontech), die
mit radioaktiv-markierten cDNAs aus den kultivierten Zellen (0.25
versus 10 ppm Zink) hybridisiert wurden. Die Arrays wurden mittels
Phosphorimager ausgelesen und die Signale auf zwei
Housekeeping-Gene (GAPDH, ß-Actin) bezogen. Die Auswertung
zeigte,
dass durch die erhöhte Zinkkonzentration ~1% der
repräsentativen Säugergene (1176) in ihrer Expression
verändert (> 1.6-fach) sind. Mittels Northern Blot-Analyse und
Real-time RT-PCR konnte für ausgewählte Gene die Modulation
der Expression verifiziert werden. Die identifizierten Gene codieren u.
a. für ein Oberflächenantigen und Proteasomproteine. Das
Genprodukt und die Funktion eines Gens (Hypothetical 40 kDa Protein)
sind bisher nicht bekannt. Das etablierte Reporterzellsystem und die
cDNA-Array-Technologie sind somit prinzipiell geeignet,
Zink-abhängige Säugergene zu identifizieren.
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Birgit
Kindermann, Hannelore Daniel, Michael W. Pfaffl
& Frank Döring (2002)
Application of the
cDNA-Array-technology for the identification of zinc response genes in
mammalian cells.
The first
international Nutrigenomics Conference, 28.2.-1.3.2002 in Noordwijk aan
Zee, The Netherlands
The
essential trace element zinc acts as a cofactor in more than 300
enzymes, it stabilizes biological membranes and it is part of the
transcriptional control of gene expression (J. Nutr. 130: 1500-1508).
Zinc deficiency in mammals causes severe metabolic disturbances and
this may in part be caused by changed expression of genes involved in
processes such as growth and development. Whereas numerous
zinc-dependent genes have been found in model organisms such as
Saccharomyces cerevisiae (PNAS 97:
7957-7962), just a few genes are known in mammals that show
zinc-dependent expression (PNAS 93: 6863-6868). We have tested the
cDNA-array-technology to identify zinc-dependent genes in a mammalian
cell system in vitro. We used the intestinal epithelial carcinoma cell
line HT29, cultured for 72 hours in media containing either a normal
zinc (0.25 ppm) or a high zinc
(10 ppm) concentration. The selected conditions caused drastic
alterations in the expression of metallothionein-1, already known as a
zinc-sensitive gene. For screening purposes we employed cDNA-arrays
(Clontech), hybridized with radioactive labeled cDNAs, which we derived
from the cultured cells (0.25 versus 10 ppm zinc). Gene expression on
the arrays was analyzed using a phosphorimager and the corresponding
signals were standardized to the housekeeping
genes GAPDH and ß-Actin. When compared to cells grown under
normal
conditions, the higher zinc concentration changed expression (> 1.6
fold) of approx. 1% of the represented mammalian genes (1176) on the
array.
Changes in the expression level of selected mRNA´s were verified
by
light-cycler PCR. Among others, a surface antigen and proteins of the
proteasom
complex showed significantly altered expression levels. Some genes have
not yet been identified by function such as a putative 40 kDa protein.
In
summary, our reporter cell system and the cDNA-array-technology appear
to be suitable for the identification of genes that show altered
expression dependent of the zinc concentration in the cell.
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Heike tom
Dieck, Hans Peter Roth, Hannelore Daniel,
Michael W. Pfaffl & Frank Döring (2002)
Expressionsanalyse von
Säugergenen beim isolierten Zinkmangel mittels cDNA und
Oligonukleotid-Arrays.
Dechema
Statusseminar „Chiptechnologie: Transkriptom – Proteom – Metabolom“, p
91
Zink ist
ein essentielles
Spurenelement und besitzt vielfältige biochemische Funktionen im
Intermediär-
und Hormonstoffwechsel sowie in der Immunabwehr. Wenngleich es als
Bestandteil
von genregulatorischen Transkriptionsfaktoren bekannt ist, sind bisher
nur
wenige zink-sensitive Gene des Säugers identifiziert worden1. Wir
haben
geprüft, inwieweit DNA-Array-Technolgie2 geeignet ist die
differentielle
Genexpression im experimentellen Zinkmangel zu erfassen. Nach Erzeugung
eines isolierten klinisch-biochemisch beschreibbaren Zinkmangels bei
Ratten
wurde deshalb für Lebergewebe eine vergleichende
Transkriptomanalyse durchgeführt. Dazu wurden zwei
DNA-Arraysysteme (Clontech, MWG-Biotech) auf der Basis von cDNA- bzw.
Oligonukleotid-Sonden eingesetzt. Die Oligonukleotid-Arrays lieferten
im Vergleich generell die reproduzierbareren Ergebnisse. Die
Genexpressionsanalyse zeigte, dass die mRNA-Spiegel von ~ 2 % der ~2500
untersuchten Gene im Zinkmangel verändert (³ 1.8-fach und
£ 0.5-fach) waren. Mittels Northernblot-Analyse und Realtime-PCR
wurden die Befunde der Arrays
bei ausgewählten Transkripten verifiziert. Signifikante
Änderungen in den Transkriptmengen ließen sich u.a. für
Wachstumsfaktor-Rezeptoren, diverse Strukturproteine, Enzyme für
Proteinmodifikationen, Enzyme des Xenobiotika-stoffwechsels und
Proteine mit Beteiligung an Exocytose-Prozessen bestimmen. Die
unterschiedlichen Funktionen der identifizierten Genprodukte
bestätigen die Annahme, dass Zink auch beim Säuger eine
Vielzahl von Genen in ihrer Expression beeinflußt. Die
auffällige Symptomatik eines isolierten Zinkmangels mit
ausgeprägter Wachtumsretardierung läßt sich also auch
auf der Ebene des Transkriptoms an Schlüsselgenen abbilden. Die
DNA-Array-Technologie könnte damit zum diagnostischen Werkzeug
für die Beurteilung der Funktion und der Versorgungslage des
Organismus mit essentiellen Mikronährstoffen werden.
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Heike tom
Dieck, Hans Peter Roth, Hannelore Daniel,
Michael W. Pfaffl & Frank Döring (2002)
Identification of genes with altered expression in zinc-deficient rats
by use of DNA microarrays.
The first
international Nutrigenomics Conference, 28.2.-1.3.2002 in Noordwijk aan
Zee, The Netherlands
The effects
of Zinc (Zn) deficieny have been established in both, humans and
experimental animals, and are known to result in anorexia, impaired
immunity, skin lesions, abnormal development und growth retardation.
Although the specific genes involved in these clinical symptoms remains
unclear. The essential trace element Zn is a constituent of hundreds of
enzymes. It also is an important component of many transcription
factors. This latter property suggests an involvement of Zn as a
regulatory ion in gene expression. Indeed, recent studies have observed
changes in transcript
level of some genes induced by Zn deficiency. With the advent of DNA
microarray technology, there is a tool which allows to study the
expression of a large number of genes simultaneously and to identify
previously unsuspected genes. In this study, we used two different
array systems to compare
the expression of more than 2500 different genes in the liver of Zn
adequate and Zn deficient rats. 32Phosphor- or fluorescence-labeled
cDNA from liver of control and Zn deficient animals were hybridized to
microarrays. By comparing gene expression profile of both groups, the
mRNA levels
of 81 genes were altered in Zn deficient status. These include genes
essential for protein modification, exocytosis, signaltransduction,
extracellular transport, structural proteins, metabolic pathways and
xenobiotic metabolism. The expression of 48 genes was reduced (< 0,5
fold), whereas the mRNA of 33 was elevated (> 1,8 fold). For 23
genes, the results were verified by northern blot analysis. 14 of these
genes were detectable on northern blots, of which 9 were confirmed as
regulated. The result indicates
that Zn deficieny causes an aberrant regulation of genes important for
growth. Further characterization of the identified genes will show
whether
they evoke the phenotype of Zn deficiency in-vivo.
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A.
STÅHLBERG1, J HÅKANSSON2, X XIAN2, H SEMB2, M. W.
PFAFFL3 P. ÅMAN4 and M. KUBISTA1
1 Department of Molecular Biotechnology, Chalmers University of
Technology and TATAA Biocenter, Sweden
2 Department of Medical Biochemistry, Gothenburg University, Sweden
3 Institute of Physiology, FML-Weihenstephan, Technical University of
Munich, Germany
4 Department of Pathology, Lundberg laboratory for cancer research,
Gothenburg University, Sweden
Advanced quantitative real-time PCR in clinical diagnostics and cDNA
microarray validation
1st European Conference in Functional Genomics and Diseases,
Prague, Czech Republic, May 14-17, 2003
Real-time PCR is the method of choice for
quantitative studies of gene expression. The method has been an
important tool in basic research for some years, and has now also
started to replace more conventional methods in clinical diagnostics.
Real-time PCR is characterized by a wide dynamic range of
quantification, high sensitivity and high precision. One of the major
problems in DNA quantification is to account for PCR inhibitions
appropriately. We have developed an in situ calibration method based on
either addition of known amount of target DNA or dilution of the test
sample to determine sample specific
PCR efficiencies. Relative gene expression in clinical samples and cDNA
microarray validations are applications particular suitable for in situ
calibration, resulting in high accuracy. In situ calibration is
particular
suitable for a few samples per gene investigations, for example: cDNA
microarray
validation and relative gene expression in clinical samples. Further,
the efficiency and reproducibility of various reverse transcription
assays
has been carefully evaluated. Our results suggest that sample to sample
variation in reverse transcription is significantly higher than in
real-time PCR, except when quantifying very low copy numbers. The
efficiency of reverse transcription differs significantly between genes
and priming strategy.
The reproducibility of reverse transcription and real-time PCR suggest
that the least difference in mRNA that can be significantly measured is
~50 % when comparing two genes in one sample and ~100 % when comparing
expression
of genes in two samples.
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