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. 

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.

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.

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.

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.

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.

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.
©  2010