Biomedical subjects

Andreas Dahl

Publications and source records attributed to Andreas Dahl.

5 recordsLinked to original sources

PubMed2007

Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips.

The analysis of gene expression is an essential element of functional genomics. Expression analysis is mainly based on DNA microarrays due to highly parallel readout and high throughput. Quantitative PCR (qPCR) based expression profiling is the gold standard for the precise monitoring of selected genes, and therefore used for validation of microarray data. Doing qPCR-based expression analysis in an array-like format can combine the higher sensitivity and accuracy of the qPCR methodology with a high data density at relatively low costs. This paper describes the development of an open-well based miniaturized platform for liquid PCR-based assays on the nanoliter scale using cost-effective polypropylene micro reactors (microPCR Chip). We show the quantification ability and reliability of qPCR in 200 nl with the microPCR chip down to 5 starting target molecules using TaqMan chemistry. An RNA expression analysis of four genes in mouse brain, liver and kidney tissues showed similar results in 200 nl as compared to standard 10 microl assays. The high sensitivity and quantification capability of the microPCR chip platform developed herein makes it a promising technology for performing high-throughput qPCR-based analysis in the nanoliter volume range.

Animals↗

PubMed2006

Matrix-assisted laser desorption/ionization mass spectrometric analysis of DNA on microarrays.

BACKGROUND: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is a powerful tool in biomolecule analysis with a wide range of application possibilities, including genotyping of single-base variations (also known as single-nucleotide polymorphisms, or SNPs) for candidate gene studies and diagnostic typing of DNA markers. We tested a method that does not require stringent purification of the nucleic acids and/or the use of modification chemistry before mass spectrometry analysis. METHODS: We used an alternative direct analysis approach that allows MALDI analysis of crude DNA samples printed on microscope slides densely coated with primary amino groups that efficiently bind negatively charged DNA. After simple washing of the slides, we applied MALDI matrix and used a conventional MALDI mass spectrometer to detect DNA products. RESULTS: We analyzed crude oligonucleotide samples and performed automated genotyping of single-base variations in 72 DNA samples. CONCLUSION: This procedure offers an operational short-cut compared with standard MALDI procedures for preparation of oligonucleotides, including purification, and thus is an efficient tool for genotyping applications, particularly those requiring accurate, flexible, and rapid data generation and medium throughput.

Amines↗

PubMed2006

Exposure to wear particles generated from studded tires and pavement induces inflammatory cytokine release from human macrophages.

Health risks associated with exposure to airborne particulate matter (PM) have been shown epidemiologically as well as experimentally, pointing to both respiratory and cardiovascular effects. Lately, wear particles generated from traffic have been recognized to be a major contributing source to the overall particle load, especially in the Nordic countries were studded tires are used. In this work, we investigated the inflammatory effect of PM10 generated from the wear of studded tires on two different types of pavement. As comparison, we also investigated PM10 from a traffic-intensive street, a subway station, and diesel exhaust particles (DEP). Human monocyte-derived macrophages, nasal epithelial cells (RPMI 2650), and bronchial epithelial cells (BEAS-2B) were exposed to the different types of particles, and the secretion of IL-6, IL-8, IL-10, and TNF-alpha into the culture medium was measured. The results show a significant release of cytokines from macrophages after exposure for all types of particles. When particles generated from asphalt/granite pavement were compared to asphalt/quartzite pavement, the granite pavement had a significantly higher capacity to induce the release of cytokines. The granite pavement particles induced cytokine release at the same magnitude as the street particles did, which was higher than what particles from both a subway station and DEP did. Exposure of epithelial cells to PM10 resulted in a significant increase of TNF-alpha secreted from BEAS-2B cells for all types of particles used (DEP was not tested), and the highest levels were induced by subway particles. None of the particle types were able to evoke detectable cytokine release from RPMI 2650 cells. The results indicate that PM10 generated by the wear of studded tires on the street surface is a large contributor to the cytokine-releasing ability of particles in traffic-intensive areas and that the type of pavement used is important for the level of this contribution. Furthermore, the airway inflammatory potential of wear particles from tires and pavement might be of a greater magnitude than that of DEP.

Base Sequence↗

PubMed2005

A chemosorptive cylindrical denuder designed for personal exposure measurements of isocyanates-evaluation on generated aerosols of 4,4'-methylenediphenyl diisocyanate.

A denuder/filter system constructed for solvent-free personal exposure measurements was evaluated for separation of vapour and particulate 4,4'-methylenediphenyl diisocyanate (4,4'-MDI) generated from heated PUR-foam. The two different phases were collected in the denuder and on the filter, respectively, by chemosorption on a polydimethylsiloxane (SE-30)-dibutylamine (DBA) stationary phase. Both repeatability and the total mass concentration of 4,4'-MDI were similar to that obtained from the reference method, in this case an impinger/filter system. The penetration of particles through the denuder at 300 ml min(-1) was nearly 100% in the particle size range 25 to 700 nm, which fits well with the Gormley-Kennedy equation. Denuder/filter sampling of the 4,4'-MDI aerosol at 500 ml min(-1) yielded a phase distribution that was in accordance with the results from the reference method. The method limit of detection was 6 ng m(-3) and 4 ng m(-3) for the denuder and filter, respectively, when using an air sampling flow rate of 300 ml min(-1) and a sampling period of 15 min. This is well below the Swedish occupational exposure limit (OEL) of 50 and 100 microg m(-3) for an 8-hour working day and a 5-min period, respectively.

Adsorption↗

PubMed2004

Automated solid-phase extraction for purification of single nucleotide polymorphism genotyping products prior to matrix-assisted laser desorption/ionisation time-of-flight mass spectrometric analysis.

In this article, we describe the application of a novel micro elution solid-phase extraction method for purification of short stretches of DNA products of single nucleotide polymorphisms (SNPs) prior to MALDI mass spectrometry analysis. An important feature of our method is that the purification columns containing a copolymer of divinylbenzene and N-vinylpyrrolidone can be used several times thereby significantly reducing costs. We implemented this DNA purification technology into a fully automated procedure including molecular biology, MALDI sample preparation, automated mass spectrometric analysis and SNP allele calling by software. Due to the facile purification protocol, the methodology shown could furthermore be used for other applications where efficient medium to high-throughput purification of nucleic acids is required.

Alkaline Phosphatase↗