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Frozen tumor tissue microarray technology for analysis of tumor RNA, DNA, and proteins

AMERICAN JOURNAL OF PATHOLOGY 2001年第5期159卷 1645-1650页

作者： Fejzo, MS Slamon, DJ Univ Calif Los Angeles Dept Med Div Hematol Oncol Sch Med Los Angeles CA 90095 USA

Tissue microarray technology is a new method used to analyze several hundred tumor samples on a single slide allowing high throughput analysis of genes and proteins on a large cohort. The original methodology involves coring tissues from paraffin-embedded tissue donor blocks and placing them into a single paraffin block. One difficulty with paraffin-embedded tissue relates to antigenic changes in proteins and mRNA degradation induced by the fixation and embedding process. We have modified this technology by using frozen tissues embedded in OCT compound as donor samples and arraying the specimens into a recipient OCT block. Tumor tissue is not fixed before embedding, and sections from the array are evaluated without fixation or postfixed according to the appropriate methodology used to analyze a specific gene at the DNA, RNA, and/or protein levels. While paraffin tissue arrays can be problematic for immunohistochemistry and for RNA in situ hybridization analyses, this method allows optimal evaluation by each technique and uniform fixation across the array panel. We show OCT arrays work well for DNA, RNA, and protein analyses, and may have significant advantages over the original technology for the assessment of some genes and proteins by improving both qualitative and quantitative results.

关键词： .slide frozen microarray technology tumor tissue paraffin Schoenberg Fejzo

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运用基因微点阵技术研究1-溴丙烷与2-溴丙烷的神经-生殖毒性(英文)

运用基因微点阵技术研究1-溴丙烷与2-溴丙烷的神经-生殖毒性(英文...

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第三届环境与职业医学国际学术研讨会

作者：李卫华丁训诚 Kitagawa Emiko Iwahashi Hitoshi 王海兰 Ichihara Sahoko Ichihara Gaku 中国上海市计划生育科学研究所·世界卫生组织人类生殖研究合作中心中国上海市计划生育科学研究所·世界卫生组织人类生殖研究合作中心日本产业技术综合研究所日本产业技术综合研究所日本名古屋大学医学部职业与环境卫生教研室日本名古屋大学医学部职业与环境卫生教研室日本名古屋大学医学部职业与环境卫生教研室

As alternatives for chlorofluorocarbons, the ozone-depleting solvents, 2-bromopropane and 1-bromopropane have been recently introduced to the workplace. In 1996,2-bromopropane was used in a Korean electronic factory and caused reproductive and hematopoietic disorders in male and female workers. This causality was revealed by animal studies and target cells were identified in subsequent *** identification of 2-bromopropane toxicity, 1-bromopropane was introduced to the workplace as a new alternative to ozone-depleting solvents. 1-bromopropane was considered less mutagenic than 2-bromopropane, but in contrast, animal experiments revealed that 1-bromopropane is a potent

关键词： 1-Biomopropane 2-Bromopropane neuro-reproductjve toxicity microarray technology

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Allergen microarray: comparison of microarray using recombinant allergens with conventional diagnostic methods to detect allergen-specific serum immunoglobulin E

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CLINICAL AND EXPERIMENTAL ALLERGY 2003年第10期33卷 1443-1449页

作者： Jahn-Schmid, B Harwanegg, C Hiller, R Bohle, B Ebner, C Scheiner, O Mueller, MW Univ Vienna Inst Pathophysiol A-1090 Vienna Austria VBC GENOMICS Biosci Res GmbH Vienna Austria

Background The availability of recombinant allergens and recent advances in biochip technology led to the development of a novel test system for the detection of allergen-specific IgE. Objective To test the performance of this allergen microarray in a serological analytical study. Methods Standard allergens contained in grass pollen (Phl p 1, Phl p 2, Phl p 5 and Phl p 6) and tree pollen ( Bet v 1 and Bet v 2) were used as a model system. The detection of allergen-specific serum IgE using microarrays was compared with standard test systems: CAP/RAST and an in-house ELISA. In order to test the analytical sensitivity of the assays, geometric dilutions of a serum pool containing high levels of pollen-specific IgE from allergic individuals were tested in each system. To assess the analytical specificity, the sera of 51 patients with presumptive allergic symptoms were collected before diagnosis. Thereafter, the results for grass/tree-pollen-specific IgE were compared. Results The microarray has a good dynamic range similar to the CAP/RAST system. microarray and ELISA showed comparable analytical sensitivity exceeding the CAP/RAST system. With respect to the analytical specificity, no significant cross-reactivity of the allergens was observed. For two of the allergens tested, weak positive signals were detected in the microarray test system, whereas they were not detectable by CAP/ RAST. Conclusion A good correlation of presently used methods to detect serum IgE and the novel microarray test system was observed. As a next step, a careful validation of this method for a multitude of allergens and a thorough clinical evaluation has to be provided. microarray testing of allergen-specific IgE can be presumed to be the method of choice for a prospective component-resolved diagnosis of Type I allergy, and the basis for the design and monitoring of a patient-tailored specific immunotherapy in the future.

关键词： IgE microarray technology recombinant allergen Type I allergy

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Developments in microarray technologies

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DRUG DISCOVERY TODAY 2003年第14期8卷 642-651页

作者： Howbrook, DN van der Valk, AM O'Shaughnessy, MC Sarker, DK Baker, SC Lloyd, AW Univ Brighton Sch Pharm & Biomol Sci Brighton BN2 4GJ E Sussex England Univ London Birkbeck Coll Sch Biol & Chem Sci London WC1E 7HX England ABgene Surrey KT19 9AP England

The focus of high-throughput drug discovery has progressed through the genome and the transcriptome and is now moving towards more difficult problems in assessing the proteome, glycome and metabolome. microarrays are currently the major tool in the assessment of gene expression via cDNA or RNA analysis;however, they are also used to screen libraries of proteins and small molecules. microarrays have helped to extract more information from smaller sample volumes and enabled the incorporation of low-cost high-throughput assays in the drug discovery process. The technology continues to develop and is being rapidly transferred into more challenging areas, with the potential to further aid and enhance the drug discovery process through the development of, for example, proteomic, glycomic and tissue arrays.

关键词： Gene Expression Profiling Nanotechnology methods Oligonucleotide Array Sequence Analysis 寡核苷酸序列分析工艺学制药 Tissue microarray Transcriptome Oligonucleotide Array Sequence Analysis Discovery Processes Gene Expression Profiling microarray technology Drug Discovery microarray RNA analysis

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Molecular screening on a compact disc

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ORGANIC & BIOMOLECULAR CHEMISTRY 2003年第18期1卷 3244-3249页

作者： La Clair, JJ Burkart, MD BIONIC BROS GmbH D-13162 Berlin Germany Univ Calif San Diego Dept Chem & Biochem La Jolla CA 92093 USA

A method is described to screen the recognition between small molecule ligands and biomolecules using a conventional compact disc ( CD) player. A procedure was developed to attach ligands to the reading face of a CD by activating the terminus of polycarbonate, a common polymer composite within the reading face of a CD. Terminal residues of the polycarbonate surface 1 were phosphorylated by reaction with ethyl-(N,N)-diisopropylamine- buffered dichloro-(N, N)-diisopropylaminophosphate to yield surface-bound chlorophosphate 2. Ligands containing a primary alcohol were condensed with 2 providing a polycarbonate capped with phosphodiester linked ligands 3 - 6. Displays were generated on the surface of a CD by printing tracks of ligands 3 - 6 on the disc with an inkjet printer. Using this method, discs were created with entire assemblies of ligand molecules distributed into separate blocks. A molecular array was developed by assembling collections of these blocks to correlate with the CDROM-XA formatted data stored within the digital layer of the disc. Regions of the disc containing a given ligand or set of ligands was marked by its spatial position using the tracking and header information. Recognition between surface expressed ligands and biomolecules was screened by an error determination routine.

关键词： CTLA4 gene NOD2 gene microarray technology Biomolecules PRINT polycarbonate ink jet printers polymer composite Compact disc players small molecule

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Biology of TiO₂-oligonucleotide nanocomposites

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NATURE MATERIALS 2003年第5期2卷 343-346页

作者： Paunesku, T Rajh, T Wiederrecht, G Maser, J Vogt, S Stojicevic, N Protic, M Lai, B Oryhon, J Thurnauer, M Woloschak, G Northwestern Univ Dept Radiol Chicago IL 60611 USA Argonne Natl Lab Biosci Div Argonne IL 60439 USA Argonne Natl Lab Div Chem Argonne IL 60439 USA Argonne Natl Lab Div Expt Facilities Argonne IL 60439 USA

Emerging areas of nanotechnology hold the promise of overcoming thelimitations of existing technologies for intracellular manipulation. THese new developments provideapproaches for the creation of chemical-biological hybrid nanocomposites that can be introduced intocells and subsequently used to initiate intracellular processes or biochemical reactions. Suchnanocomposites would advance medical biotechnology, just as they are improving microarray technologyand imaging in biology and medicine, and introducing new possibilities in chemistry and materialsciences. Here we describe the behaviour of 45-A nanoparticles of titanium dioxide semiconductorcombined with oligonucleotide DNA into nanocomposites in vivo and in vitro. These nanocomposites notonly retain the intrinsic photocatalytic capacity of TiO_2 and the bioactivity of theoligonucleotide DNA (covalently attached to the TiO_2 nanoparticle), but also possess the chemicallyand biologically unique new property of a light-inducible nucleic acid endonuclease, which couldbecome a new tool for gene therapy.

关键词： Genetic Therapy Oligonucleotides luminescent characteristics Nanotechnologies Nanocomposites Biological Science Disciplines microarray technology Medicine materials science biology Biological activity

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Gene expression in two hepatic cell lines, cultured primary hepatocytes, and liver slices compared to the in vivo liver gene expression in rats:: Possible implications for toxicogenomics use of in vitro systems

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TOXICOLOGICAL SCIENCES 2003年第2期73卷 386-402页

作者： Boess, F Kamber, M Romer, S Gasser, R Muller, D Albertini, S Suter, L F Hoffmann La Roche & Co Ltd Div Pharmaceut Nonclin Dev PRNS CH-4070 Basel Switzerland Basilea Pharmaceut Ltd CH-4002 Basel Switzerland Univ Jena Inst Pharmacol & Toxicol D-07743 Jena Germany

microarray technology allows the simultaneous analysis of mRNA expression levels of thousands of genes. In the field of toxicogenomics, this technology could help to identify potentially unsafe compounds based on the changes in mRNA expression patterns they induce. Rodent in vivo and in vitro systems are currently the experimental models of choice for predictive toxicology, especially in early phases of development. This study characterizes several hepatic in vitro systems based on mRNA expression profiles, comparing them to gene expression in liver tissue. The in vitro systems investigated comprise two rat liver cell lines (BRL3A and NRL clone 9), primary hepatocytes in conventional monolayer or in sandwich culture, and liver slices. The results demonstrate that liver slices exhibit the strongest similarity to liver tissue regarding mRNA expression, whereas the two cell lines are quite different from the whole liver. We were able to identify genes with strong changes in expression levels in all or at least one of the in vitro systems relative to whole liver. In particular, for some cytochrome P450s the differences observed on the mRNA expression level were paralleled by protein expression and enzymatic activity. In addition, the effect of time in culture was assessed. We were able to show a profound effect of the duration of culture. Expression patterns change most rapidly soon after cell isolation and culture initiation and stabilize with time in culture. The findings are discussed with respect to the usefulness of the various hepatic in vitro systems for microarray-based toxicological testing of compounds.

关键词： microarray technology hepatic in vitro system rat liver cell line sandwich culture monolayer liver slice

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microarrayed recombinant allergens for diagnosis of allergy

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CLINICAL AND EXPERIMENTAL ALLERGY 2003年第1期33卷 7-13页

作者： Harwanegg, C Laffer, S Hiller, R Mueller, MW Kraft, D Spitzauer, S Valenta, R Univ Vienna Sch MedVienna Gen Hosp Dept Pathophysiol Mol Immunopathol Grp A-1090 Vienna Austria Univ Vienna Vienna Gen Hosp Clin Inst Med & Chem Lab Diagnost A-1090 Vienna Austria VBC Genom Biosci Res GmbH Vienna Austria

We suggest that the coapplication of recombinant allergens and microarray technology can lead to the development of new forms of multi-allergen tests which allow the determining and monitoring of complex sensitization profiles of allergic patients in single assays. The allergen extracts which have so far been used for diagnosis only allowed the determining of whether an allergic patient is sensitized against a particular allergen source, but the disease-eliciting allergens could not be identified. Through the application of recombinant DNA technology a rapidly growing panel of recombinant allergen molecules has become available which meanwhile comprises the epitope spectrum of most of the important allergen sources. We demonstrate that microarray technology can be used to establish multi-allergen tests consisting of microarrayed recombinant allergen molecules. microarrayed recombinant allergens can be used to determine and monitor the profile of disease-eliciting allergens using single tests that require minute amounts of serum from allergic patients. The wealth of diagnostic information gained through microarray-based allergy testing will likely improve diagnosis, prevention and treatment of allergy.

关键词： Allergens Diagnostic Hypersensitivity microarray technology Unmarried

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Array rank order regression analysis for the detection of gene copy-number changes in human cancer

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GENOMICS 2003年第2期82卷 122-129页

作者： Cheng, C Kimmel, R Neiman, P Zhao, LP Fred Hutchinson Canc Res Ctr Div Publ Hlth Sci Seattle WA 98109 USA Fred Hutchinson Canc Res Ctr Div Basic Sci Seattle WA 98109 USA

cDNA microarray technology has been applied to the detection of DNA copy-number changes in malignant tumors. Test and control genomic DNA samples are differentially labeled and cohybridized to a spotted cDNA microarray. The ratio of test to control fluorescence intensities for each spot reflects relative gene copy number. The low signal-to-noise ratios of this assay and the variable levels of gene amplification and deletion among tumors hamper the detection of deviations from the diploid complement. We describe a regression-based statistical method to test for altered copy number on each gene and apply the technique to copy-number profiles in 10 thyroid tumors. We show that a novel transformation of fluorescence ratios into array rank order efficiently normalizes the heterogeneity among copy-number profiles and improves the reproducibility of the results. Array rank order regression analysis enhances the detection of consistent changes in gene copy number in solid tumors by cDNA microarray-based comparative genome hybridization. (C) 2003 Elsevier Science (USA). All rights reserved.

关键词： CHG microarray technology rank order regression analysis statistics

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Activation of the protein kinase A pathway in human endometrial stromal cells reveals sequential categorical gene regulation

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PHYSIOLOGICAL GENOMICS 2003年第1期16卷 47-66页

作者： Tierney, EP Tulac, S Huang, STJ Giudice, LC Stanford Univ Sch Med Dept Gynecol & Obstet Div Reprod Endocrinol & Infertil Stanford CA 94305 USA

Decidualization of endometrial stromal cells is a prerequisite for human implantation and occurs in vivo in response to progesterone and involves activation of the protein kinase A ( PKA) pathway. The objective of this study was to determine the molecular signatures and patterns of gene expression during stimulation of this pathway with an analog of cAMP. Endometrial stromal cells from two subjects were treated with or without 8- Br- cAMP ( 1 mM) for 0, 2, 12, 24, 36, and 48 h and were processed for microarray analysis, screening for 12,686 genes and ESTs. Most abundantly upregulated genes included neuropeptides, immune genes, IGF family members, cell cycle regulators, extracellular matrix proteases, cholesterol trafficking, cell growth and differentiation, hormone signaling, and signal transduction. Most abundantly downregulated genes included activator of NF- kappa B, actin/ tropomyosin/ calmodulin binding protein, cyclin B, IGFBP- 5, alpha1 type XVI collagen, lipocortin III, L- kynurenine hydrolase, frizzle- related protein, and cyclin E2. RT- PCR validated upregulation of IGFBP- 1, preprosomatostatin, and IL- 11, and Northern analysis validated their kinetic upregulation. RT- PCR confirmed downregulation of IGFBP- 5, cyclin B, and TIL- 4. K- means analysis revealed four major patterns of up- and downregulated genes, and genes within each ontological group were categorized into these four kinetic patterns. Within each ontological group different patterns of temporal gene expression were observed, indicating that even genes within one functional category are regulated differently during activation of the PKA pathway in human endometrial stromal cells. Overall, the data demonstrate kinetic reprogramming of genes within specific functional groups and changes in genes associated with nucleic acid binding, cell proliferation, decreased G protein signaling, increased STAT pathway signaling, structural proteins, cellular differentiation, and secretory processes. These c

关键词： endometrium decidualization implantation microarray technology

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