Uwe R. Müller

Vitamin D growth inhibition of breast cancer cells: gene expression patterns assessed by cDNA microarray.

Abstract1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth. Although it is evident that 1,25(OH)2D3 inhibits growth of both estrogen receptor alpha-positive [ERα(+)] and -negative [ERα(−)] breast cancer cells, the cellular pathways contributing to these effects remain unclear. We studied the gene expression patterns in ERα(+) MCF-7 and ERα(−) MDA MB 231 human breast cancer cells following 1,25(OH)2D3 treatment, using cDNA expression arrays. Both cell lines showed a significant induction of the 1,25(OH)2D3-dependent 24-hydroxylase gene, a marker for the actions of 1,25(OH)2D3. In MCF-7 cells, 51 genes were up-regulated and 19 genes were down-regulated. The up-regulated genes encoded cell adhesion molecules, growth factors/modulators, steroid receptors/co-activators, cytokines, kinases and transcription factors. Of the up-regulated genes, 40% were implicated in cell cycle regulation and apoptosis and included cyclin G1 and cyclin I, p21-activated kinase-1 (PAK-1), p53, retinoblastoma like-2 [Rb2 (p130)], insulin-like growth factor binding protein-5 (IGFBP5) and caspases. Among the down-regulated genes were ERα, growth factors, cytokines and several kinases. Some of these results were confirmed by real-time PCR. In MDA MB 231 cells, 20 genes were up-regulated and 13 genes were down-regulated. Very few genes directly implicated in cell cycle regulation were up-regulated. The matrix metalloproteinases formed a major class of genes that were down-regulated in the MDA MB 231 cells. Seven genes were commonly up-regulated in both cell lines and these included transforming growth factor (TGFβ2) and Rb2 (p130). In conclusion, the gene expression profiles of the two cell lines studied were different with a few overlapping genes suggesting that different cellular pathways might be regulated by 1,25(OH)2D3 to exert its growth inhibitory effects in ERα(+) and ERα(−) cells.

SNP identification in unamplified human genomic DNA with gold nanoparticle probes

Single nucleotide polymorphisms (SNPs) comprise the most abundant source of genetic variation in the human genome. SNPs may be linked to genetic predispositions, frank disorders or adverse drug responses, or they may serve as genetic markers in linkage disequilibrium analysis. Thus far, established SNP detection techniques have utilized enzymes to meet the sensitivity and specificity requirements needed to overcome the high complexity of the human genome. Herein, we present for the first time a microarray-based method that allows multiplex SNP genotyping in total human genomic DNA without the need for target amplification or complexity reduction. This direct SNP genotyping methodology requires no enzymes and relies on the high sensitivity of the gold nanoparticle probes. Specificity is derived from two sequential oligonucleotide hybridizations to the target by allele-specific surface-immobilized capture probes and gene-specific oligonucleotide-functionalized gold nanoparticle probes. Reproducible multiplex SNP detection is demonstrated with unamplified human genomic DNA samples representing all possible genotypes for three genes involved in thrombotic disorders. The assay format is simple, rapid and robust pointing to its suitability for multiplex SNP profiling at the ‘point of care’.

Rare earth-doped glass microbarcodes

The development of ultraminiaturized identification tags has applications in fields ranging from advanced biotechnology to security. This paper describes micrometer-sized glass barcodes containing a pattern of different fluorescent materials that are easily identified by using a UV lamp and an optical microscope. A model DNA hybridization assay using these “microbarcodes” is described. Rare earth-doped glasses were chosen because of their narrow emission bands, high quantum efficiencies, noninterference with common fluorescent labels, and inertness to most organic and aqueous solvents. These properties and the large number (>1 million) of possible combinations of these microbarcodes make them attractive for use in multiplexed bioassays and general encoding.

Microfluidic devices for fluidic circulation and mixing improve hybridization signal intensity on DNA arrays

Reactions of biomolecules with surface mounted materials on microscope slides are often limited by slow diffusion kinetics, especially in low volumes where diffusion is the only means of mixing. This is a particular problem for reactions where only small amounts of analyte are available and the required reaction volume limits the analyte concentration. A low volume microfluidic device consisting of two interconnected 9 mm x 37.5 mm reaction chambers was developed to allow mixing and closed loop fluidic circulation over most of the surface of a microscope slide. Fluid samples are moved from one reaction chamber to the other by the rotation of a magnetic stirring bar that is driven by a standard magnetic stirrer. We demonstrate that circulation and mixing of different reagents can be efficiently accomplished by this closed loop device with solutions varying in viscosity from 1 to 16.2 centipoise. We also show by example of a microarray hybridization that the reaction efficiency can be enhanced 2-5 fold through fluid mixing under conditions where diffusion is rate limiting. For comparison, similar results were achieved with a disposable commercial device that covers only half of the reaction area of the closed loop device.

Diagnostic Detection Systems Based on Gold Nanoparticle Probes

The development of a nanoparticle based detection methodology for DNA microarray applications is described. Originally reported by Mirkin and co-workers, the technology utilizes gold nanoparticles derivatized with thiol-modified oligonucleotides that are designed to bind complementary DNA targets. A glass surface with arrays of immobilized DNA capture sequences is used to capture DNA targets, which are then detected via hybridization to the gold nanoparticle probes. For maximum signal strength a layer of silver is deposited onto the gold nanoparticles, providing for highly sensitive and specific detection of target sequences using low cost optical detection systems. The relative optical detection limits for silver amplified gold nanoparticle probes and Cy3 based fluorescence have been tested. Furthermore, we report progress towards the direct detection of non-amplified genomic DNA from infectious agents and single nucleotide polymorphisms (SNP) in human genomic DNA based on nanoparticle technology.

Microarrays and DNA-Chips in the Analysis of Genetic Aberrations

Fluorescence in situ hybridization (FISH) is a precise technique to determine copy numbers of a particular sequence in individual cells with a resolution better than 30 Kb, but requires a priori knowledge of the target sequence to be analyzed. Comparative genomic hybridization (CGH) on the other hand allows a global analysis of the whole genome, but at significant loss in resolution (on the order of 10–15 Mb). DNA chip technology (so called genosensor-based or matrix-based CGH) combines the high resolution of FISH with the inherent multiplex capability of CGH, and adds simplicity to the analysis. This technology represents the latest addition to chip based genetic analyses.

Microarray-based STR genotyping using RecA-mediated ligation.

We describe a novel assay capable of accurately determining the length of short tandem repeat (STR) alleles. STR genotyping is achieved utilizing RecA-mediated ligation (RML), which combines the high fidelity of RecA-mediated homology searching with allele-specific ligation. RecA catalyzes the pairing of synthetic oligonucleotides with one strand of a double-stranded DNA target, in this case a PCR amplicon. Ligation occurs only when two adjacent oligonucleotides are base paired to the STR region without any overlap or gap. RecA activity is required to overcome the inherent difficulty of annealing repeated sequences in register. This assay is capable of determining STR genotypes of human samples, is easily adapted to high throughput or automated systems and can have widespread utility in diagnostic and forensic applications.

High Sensitivity Expression Profiling

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Novel Surface Technologies for Genomics, Proteomics, and Drug Discovery

Following the recent progress in functional genomics and proteomics, and high-throughput screening (HTS) in drug discovery, evolving technologies over the last decade have offered a tremendous leap over the caveats of traditional techniques. In response to this metamorphosis of technologies through different platforms, Corning has introduced a suite of surface technologies with applications in microarray printing, enhanced attachment, and consumables in drug discovery. Microarrays generated on an ultra-flat glass substrate with GAPS coating exhibiting a robust chemistry and low surface background have led to higher sensitivity and reproducibility for the expression assay. Recent introduction of UltraGAPS™ surface enables oligo attachment for use in differential gene expression analysis. Various attachment surfaces to meet the needs of the applications in genomics, proteomics and drug discovery will be discussed.