Biomedical subjects

Meinhard Hahn

Publications and source records attributed to Meinhard Hahn.

9 recordsLinked to original sources

PubMed2003

Increased expression of disintegrin-metalloproteinases ADAM-15 and ADAM-9 following upregulation of integrins alpha5beta1 and alphavbeta3 in atherosclerosis.

Regulation of alphavbeta3 and alpha5beta1 integrin function plays a crucial role in atherosclerosis. Possible regulators of integrin-matrix interactions are integrin-binding ADAMs (proteins with a disintegrin- and metalloproteinase-domain), like ADAM-15 and ADAM-9. Molecular interactions between ADAM-15, alpha5beta1, and alphavbeta3 have been demonstrated. ADAM-9 and ADAM-15 were found to be interdependently regulated. This study, therefore, investigated whether the upregulation of integrins alpha5beta1 and alphavbeta3 was correlated with the expression of integrin-binding ADAMs in atherosclerotic processes. Human arterial and venous vascular smooth muscle cells (VSMCs) were incubated with PDGF over different time intervals up to a 3-day culture period. mRNA concentrations, quantified by real-time RT-PCR and normalized to PBGD, of integrins alphavbeta3 and alpha5beta1 were strongly increased after a 12-h PDGF-incubation in arterial and venous VSMC. ADAM-15 and ADAM-9 mRNA production showed a corresponding increase following integrin upregulation after a 24-h incubation period. Western blot anaylsis revealed an increased protein expression of integrins and ADAMs in PDGF-stimulated VSMC. Additionally, mRNA concentrations of atherosclerotic and normal human specimens were quantified by real-time RT-PCR. mRNA of ADAMs and integrins was significantly increased in atherosclerotic arteries compared to normal arteries. Immunohistochemistry of these specimens showed an increased expression and codistribution of both ADAMs and integrins in atherosclerosis. In conclusion, upregulation of ADAM-15 and ADAM-9 in atherosclerosis appears to follow an increase in alpha5beta1 and alphavbeta3 integrins. Since alpha5beta1 and alphavbeta3 are known to promote smooth muscle cell migration and proliferation, upregulation of ADAM-15 and ADAM-9 could balance integrin-matrix interactions and cell migration, thus modulating neointima progression.

ADAM Proteins↗

PubMed2003

Validation of an algorithm for automatic quantification of nucleic acid copy numbers by real-time polymerase chain reaction.

Real-time quantitative polymerase chain reaction (PCR) with on-line fluorescence detection has become an important technique not only for determination of the absolute or relative copy number of nucleic acids but also for mutation detection, which is usually done by measuring melting curves. Optimum assay conditions have been established for a variety of targets and experimental setups, but only limited attention has been directed to data evaluation and validation of the results. In this work, algorithms for the processing of real-time PCR data are evaluated for several target sequences (p53, IGF-1, PAI-1, Factor VIIc) and compared to the results obtained by standard procedures. The algorithms are implemented in software called SoFAR, which allows fully automatic analysis of real-time PCR data obtained with a Roche LightCycler instrument. The software yields results with considerably increased precision and accuracy of quantifications. This is achieved mainly by the correction of amplification-independent signal trends and a robust fit of the exponential phase of the signal curves. The melting curve data are corrected for signal changes not due to the melting process and are smoothed by fitting cubic splines. Therefore, sensitivity, resolution, and accuracy of melting curve analyses are improved.

Algorithms↗

PubMed2003

Optimization of high-density cDNA-microarray protocols by 'design of experiments'.

Expression analysis using microarray technology implies a complex experimental procedure with a large number of parameters affecting the final result. We have demonstrated that optimization of such a complex protocol can be far better handled using design of experiments (DOE) than by working on a single parameter at a time. Based on the results of a screening design, we developed a spotting buffer composed of formamide, betaine and nitrocellulose. This buffer provides a 2-fold increase in signal-to-background ratio compared to 3x SSC. Comparison to seven other buffers tested on 10 different substrates revealed it had the highest sensitivity. DNA dissolved in this buffer can be spotted on epoxysilane-coated microscope slides at a density of up to 70 000 spots per slide. A second DOE approach characterized the RNA labeling process with regard to the concentration of fluorescent dyes, dNTPs and reverse transcriptase. Adjust ments of the concentrations of dNTPs, as well as reverse transcriptase, towards the optimum, produced an improvement in the performance of the labeling procedure by a factor of 3 (Cy3) and 10 (Cy5). These results demonstrate that the process of establishing a stable expression profiling protocol and its further optimization can be significantly shortened and improved by DOE.

Buffers↗

PubMed2003

Real-time PCR-based method for the estimation of genome sizes.

The fast and reliable estimation of the genome sizes of various species would allow for a systematic analysis of many organisms and could reveal insights into evolutionary processes. Many methods for the estimation of genome sizes have already been described. The classical methods are based on the determination of the phosphate content in the DNA backbone of total DNA isolated from a defined number of cells or on reassociation kinetics of high molecular weight genomic DNA (c(0)t assay). More recent techniques employ DNA-specific fluorescent dyes in flow cytometry analysis, image analysis or absorption cytometry after Feulgen staining. The method presented here is based on the absolute quantification of genetic elements in a known amount (mass) of genomic DNA by real-time quantitative PCR. The method was evaluated on three different eukaryotic species, Saccharomyces cerevisiae (12.1 Mb), Xiphophorus maculatus (550 Mb) and Homo sapiens sapiens (2.9 Gb), and found to be fast, highly accurate and reliable.

Animals↗

PubMed2003

Profile of gene expression induced by the tumour promotor TPA in murine epithelial cells.

Malignant transformation of mouse skin by chemical carcinogens and tumour promoters, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), is a multistage process that leads to squamous cell carcinoma (SCC) formation. In an effort to identify tumour-associated genes, we studied the influence of short-term TPA-treatment on the gene expression profile of murine skin. A comprehensive microarray with some 5,000 murine gene specific cDNA fragments was established and hybridised with pooled RNA derived from control and TPA-treated dorsal skin samples. Of these genes, 54 were up- and 35 were down-regulated upon TPA application. Additionally, we performed suppression subtractive hybridisation (SSH) with respective RNA pools to generate and analyse a cDNA library enriched for TPA-inducible genes. Expression data of selected genes were confirmed by quantitative real-time PCR and Northern blot analysis. Comparison of microarray and SSH data revealed that 26% of up-regulated genes identified by expression profiling matched with those present in the SSH library. Besides numerous known genes, we identified a large set of unknown cDNAs that represent previously unrecognised TPA-regulated genes in murine skin with potential function in tumour promotion. Additionally, some TPA-induced genes, such as Sprr1A, Saa3, JunB, Il4ralpha, Gp38, RalGDS and Slpi exhibit high basal level in advanced stages of skin carcinogenesis, suggesting that at least a subgroup of the identified TPA-regulated genes may contribute to tumour progression and metastasis.

Animals↗

PubMed2003

QuickLIMS: facilitating the data management for DNA-microarray fabrication.

SUMMARY: QuickLIMS is a Microsoft Access-based laboratory information and management system, capable of processing all information for microarray production. The program's operational flow is protocol-based, dynamically adapting to changes of the process. It interacts with the laboratory robot and with other database systems over the network, and it represents a complete solution for the management of the entire manufacturing process. AVAILABILITY AND SUPPLEMENTARY INFORMATION: http://www.dkfz-heidelberg.de/kompl_genome/Other/QuickLims/index.html

Computer Communication Networks↗

PubMed2003

HER-2/neu gene copy number quantified by real-time PCR: comparison of gene amplification, heterozygosity, and immunohistochemical status in breast cancer tissue.

BACKGROUND: Amplification of the oncogene HER-2/neu influences breast cancer pathogenesis, and therapy and prognosis may be affected by the degree of amplification. The extent of amplification or protein overexpression typically is analyzed by fluorescence in situ hybridization or immunohistochemistry (IHC), but quantitative PCR techniques have been described that may provide alternatives to these methods. METHODS: We developed a rapid-cycle, real-time PCR assay for quantification of HER-2/neu gene status. We compared results obtained with this assay with short tandem repeat findings by capillary electrophoresis (CE) and with protein overexpression assessments by IHC. Accuracy and linearity were tested on cell lines and with simulation experiments. We analyzed the amplification of HER-2/neu in 51 clinical tissue samples from patients with suspected breast cancer. RESULTS: The intra- and interrun CVs for HER-2/neu quantification by real-time PCR were 12% and 18%, and the CV for different simulated amplification and deletion experiments was <7%. The results for HER-2/neu gene status in cell lines matched the values reported in literature. We detected HER-2/neu amplification by real-time PCR in 11 samples, all from patients with invasive ductal carcinoma. Allelic imbalances were found by CE analyses in three samples and by protein overexpression in six samples; five of these were also detected by real-time PCR. Comparison of the quantification results with known prognostic indices yielded results similar to those reported in several other published studies. CONCLUSIONS: The assay is suitable for accurate and precise quantification of HER-2/neu copy numbers in tumor tissue samples obtained in routine clinical practice.

Adult↗

PubMed2003

SoFAR: software for fully automatic evaluation of real-time PCR data.

Quantitative real-time PCR has proven to be an extremely useful technique in life sciences for many applications. Although a lot of attention has been paid to the optimization of the assay conditions, the analysis of the data acquired is often done with software tools that do not make optimum use of the information provided by the data. Particularly, this is the case for high-throughput analysis, which requires a careful characterization and interpretation of the complete data by suitable software. Here we present a software solution for the robust, reliable, accurate, and fast evaluation of real-time PCR data, called SoFAR. The software automatically evaluates the data acquired with the LightCycler system. It applies new algorithms for an adaptive background correction of signal trends, the calculation of the effective signal noise, the automated identification of the exponential phases, the adaptive smoothing of the raw data, and the correction of melting curve data. Finally, it provides information regarding the validity of the results obtained. The SoFAR software minimizes the time required for evaluation and increases the accuracy and reliability of the results. The software is available upon request.

Algorithms↗

PubMed2002

Detection and quantification of insertion/deletion variations by allele-specific real-time PCR: application for genotyping and chimerism analysis.

The DNA-based quantitative analysis of genetic chimerism is becoming increasingly more important for molecular biology in general and molecular medicine in particular. Useful genomic targets for these analyses are polymorphic sequences, but here the problem of a reliable quantification with high dynamic range is not yet satisfactorily solved. To this end we have combined the allele-specific amplification with a real-time PCR-based quantification for rapid allelotyping and chimerism analysis. The sequence variations are discriminated by the 3'-end of the allele-specific primer. Amplification is monitored by SYBR-Green I fluorescence. We demonstrate the efficiency of this method for two clinically relevant targets: (i) the 10 bp insertion/deletion polymorphism in the promoter of the factor VIIc (F-VIIc) gene and (ii) the 4G/5G single nucleotide polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene. Both polymorphisms are associated with clinical risk factors. Allelotyping results were in complete agreement with those obtained by reference methods. Mixed chimeric DNA samples could be quantified reliably with a dynamic range of 1:3000 for an easy target (F-VIIc) and of 1:64 for a difficult target (PAI-1). Our protocol is particularly useful for rapid, reliable and inexpensive genotyping and quantitative chimerism analysis without requiring expensive fluorophor dye labelled probes.

Alleles↗