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Microarray Platform (microarray + platform)
Selected AbstractsCell microarray platform for anticancer drug development,DRUG DEVELOPMENT RESEARCH, Issue 5 2007Min-Jung Lee Abstract Pharmacodynamic assessment of whether a drug has interacted with and modified its target is an essential component of molecularly targeted clinical trials. Although many trials are written with the intent to assess tumor biopsies, if available, thus far the great majority of early drug trials have used peripheral blood mononuclear cells (PBMC) as a tumor surrogate. Typically, PBMC are studied by low-throughput techniques such as Western blot. We present the use of a cell-based tissue microarray for assessment of anticancer drug activity in vivo. We demonstrate the utility of this technique for analysis of protein hyperacetylation in response to treatment with the histone deacetylase inhibitor, SNDX-275 in PBMC treated in vitro and in PBMC and bone marrow aspirates from patients in Phase I clinical trials with SNDX-275. We demonstrate that the cell microarray can be used to measure drug response in a high-throughput manner, allowing analysis of an entire trial on one or two glass slides. The cell microarray technique brings the advantages of the tissue microarray platform to the pharmacodynamic assessment of single cells, such as those isolated from bone marrow aspirates, fine needle aspirates, or malignant effusions, and to analysis of PBMC, the most commonly studied surrogate in oncology trials. Drug Dev Res 68:226,234, 2007. Published 2007 Wiley-Liss, Inc. [source] Development and application of the human intestinal tract chip, a phylogenetic microarray: analysis of universally conserved phylotypes in the abundant microbiota of young and elderly adultsENVIRONMENTAL MICROBIOLOGY, Issue 7 2009-Stojanovi, Mirjana Rajili Summary In this paper we present the in silico assessment of the diversity of variable regions of the small subunit ribosomal RNA (SSU rRNA) gene based on an ecosystem-specific curated database, describe a probe design procedure based on two hypervariable regions with minimal redundancy and test the potential of such probe design strategy for the design of a flexible microarray platform. This resulted in the development and application of a phylogenetic microarray for studying the human gastrointestinal microbiota , referred as the human intestinal tract chip (HITChip). Over 4800 dedicated tiling oligonucleotide probes were designed based on two hypervariable regions of the SSU rRNA gene of 1140 unique microbial phylotypes (< 98% identity) following analysis of over 16 000 human intestinal SSU rRNA sequences. These HITChip probes were hybridized to a diverse set of human intestinal samples and SSU rRNA clones to validate its fingerprinting and quantification potential. Excellent reproducibility (median Pearson's correlation of 0.99) was obtained following hybridization with T7 polymerase transcripts generated in vitro from SSU rRNA gene amplicons. A linear dose,response was observed with artificial mixtures of 40 different representative amplicons with relative abundances as low as 0.1% of total microbiota. Analysis of three consecutively collected faecal samples from ten individuals (five young and five elderly adults) revealed temporal dynamics and confirmed that the adult intestinal microbiota is an individual-specific and relatively stable ecosystem. Further analysis of the stable part allowed for the identification of a universal microbiota core at the approximate genus level (90% sequence similarity). This core consists of members of Actinobacteria, Bacteroidetes and Firmicutes. Used as a phylogenetic fingerprinting tool with the possibility for relative quantification, the HITChip has the potential to bridge the gaps in our knowledge in the quantitative and qualitative description of the human gastrointestinal microbiota composition. [source] Detection of bacteria associated with harmful algal blooms from coastal and microcosm environments using electronic microarraysENVIRONMENTAL MICROBIOLOGY, Issue 3 2007Edward A. Barlaan Summary With the global expansion of harmful algal blooms (HABs), several measures, including molecular approaches, have been undertaken to monitor its occurrence. Many reports have indicated the significant roles of bacteria in controlling algal bloom dynamics. Attempts have been made to utilize the bacteria/harmful algae relationship in HAB monitoring. In this study, bacterial assemblages monitored during coastal HABs and bacterial communities in induced microcosm blooms were investigated. Samples were analysed using denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA gene. DGGE bands with peculiar patterns before, during, and after algal blooms were isolated and identified. Probes for six ribotypes representing organisms associated with Chatonella spp., Heterocapsa circularisquama, or Heterosigma akashiwo were used for analysis on NanoChip electronic microarray. In addition, a new approach using cultured bacteria species was developed to detect longer (533 bp) polymerase chain reaction-amplified products on the electronic microarray. The use of fluorescently labelled primers allowed the detection of individual species in single or mixed DNA conditions. The developed approach enabled the detection of the presence or absence and relative abundance of the HAB-related ribotypes in coastal and microcosm blooms. This study indicates the ability of electronic microarray platform to detect or monitor bacteria in natural and induced environments. [source] Transcriptional Analysis of Buffalo (Bubalus bubalis) Oocytes During In Vitro Maturation Using Bovine cDNA MicroarrayREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010OM Kandil Contents The need for improving in vitro production of buffalo embryos necessitates a better understanding of the molecular mechanisms regulating early development including oocyte maturation. Here, we used bovine cDNA microarray platform to investigate mRNA abundance of buffalo oocytes before and after in vitro maturation. For this, a total of six pools each contains 50 immature or in vitro matured buffalo oocytes were used for mRNA isolation and subsequent cDNA synthesis. The BlueChip bovine cDNA microarray (with approximately 2000 clones) was used to analyse gene expression profiles between immature and matured oocytes. Statistical analysis of microarray data revealed a total of 104 transcripts to be differentially expressed between the two oocyte groups. Among these, transcription factors (ZFP91), M-phase mitotic cell cycle (MPHOSPH9), growth factor (BMP15) and DNA binding (HMGN2) were found to be up-regulated in immature oocytes. Similarly, matured oocytes were found to be enriched with genes involved in cytoskeleton (ACTB), hydrogen ion transporting (ATP6V1C2) and structural constituent of ribosome (RPS27A). Quantitative real-time polymerase chain reaction validated the expression profile of some selected transcripts during array analysis. In conclusion, to our knowledge, this is the first large-scale expression study to identify candidate genes differentially abundant and with potential role during buffalo oocyte maturation. [source] Salinity stress adaptation competence in the extremophile Thellungiella halophila in comparison with its relative Arabidopsis thalianaTHE PLANT JOURNAL, Issue 5 2005Qingqiu Gong Summary In stark contrast to Arabidopsis, a related species, Thellungiella halophila (Thellungiella salsuginea; salt cress), displays extreme tolerance to high salinity, low humidity and freezing. High nucleotide sequence identity permits the use of tools developed for Arabidopsis for Thellungiella transcript profiling, for which a microarray platform with >25 000 DNA elements (70-mer oligonucleotides) was used. Microarray transcript profiling and intensity analysis, quantitative RT-PCR, and metabolite profiles define genes and pathways that showed shared and divergent responses to salinity stress in the two species. Shared responses are exemplified by 40% of the regulated genes functioning in confining ribosomal functions, photosynthesis and cell growth, as well as activating osmolyte production, transport activities and abscisic acid-dependent pathways. An additional 60% of regulated genes distinguished Thellungiella from Arabidopsis. Analysis of the differences showed that Arabidopsis exhibited a global defense strategy that required bulk protein synthesis, while Thellungiella induced genes functioning in protein folding, post-translational modification and protein redistribution. At 150 mm NaCl, Thellungiella maintained unimpeded growth. Transcript intensity analyses and metabolite profiles supported the microarray results, pointing towards a stress-anticipatory preparedness in Thellungiella. [source] Using microarray technology to select housekeeping genes in Chinese hamster ovary cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2009Scott M. Bahr Abstract In the present study, we have identified species-specific housekeeping genes (HKGs) for Chinese Hamster Ovary (CHO) cells using data from microarray gene expression profiling. HKGs suitable for quantitative RT-PCR normalization should display relatively stable expression levels across experimental conditions. We analyzed transcription profiles of several IgG-producing recombinant CHO cell lines under numerous culture conditions using a custom CHO DNA microarray platform and calculated relative expression variability from 124 microarrays. We selected a novel panel of candidate HKGs based on their low variability in expression from the microarray data. Compared to three traditional HKGs (Gapdh, Actb, and B2m), the majority of genes on this panel demonstrated lower or equal variability. Each candidate HKG was then validated using qRT-PCR. Final selection of CHO-specific HKGs include Actr5, Eif3i, Hirip3, Pabpn1, Vezt, Cog1, and Yaf2. The results reported here provide a useful tool for gene expression analyses in CHO cells, a critical expression platform used in biotherapeutics. Biotechnol. Bioeng. 2009; 104: 1041,1046. © 2009 Wiley Periodicals, Inc. [source] Heterogeneity of gene expression profiles in head and neck cancerHEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 12 2007Jimmy Pramana MD Abstract Background. Results of gene expression profiling studies from different institutes often lack consistency. This could be due to the use of different microarray platforms and protocols, or to intratumoral heterogeneity in mRNA expression. The aim of our study was to quantify intratumoral heterogeneity in head and neck cancer. Methods. Forty-four fresh frozen biopsies were taken from 22 patients, 2 per tumor. RNA was extracted, tested for quality, amplified, labeled, and hybridized to a 35k oligoarray. Results. Unsupervised clustering analyses using all genes, the most variable genes, or random gene sets showed that 80% to 90% of biopsy pairs clustered together. A within-pair-between-pair scatter ratio analysis showed that the similarity between matching pairs was significantly greater than that between random pairs (p <.00001). Conclusions. Two biopsies from the same tumor show far greater similarity in gene expression than biopsies from different tumors, supporting the use of 1 biopsy for expression profiling. © 2007 Wiley Periodicals, Inc. Head Neck 2007 [source] Analysis of several fluorescent detector molecules for protein microarray useLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 1 2003Rick Wiese Abstract The utility of several streptavidin-linked fluorescent detector molecules was evaluated on two protein microarray platforms. Tested detector molecules included: Alexa Fluor 546; R-phycoerythrin (RPE), orange fluospheres; Cy3-containing liposomes (Large Unilamellar Vesicles, LUV) labelled with Cy3; and an RPE,antibody complex. The two array architectures tested consisted of an array of murine Fc,biotin and an array of murine IgG (the murine IgG array was probed with a biotinylated rabbit anti-murine IgG). These platforms allowed for the direct comparison of detector utility by detector recognition of array-bound biotin. All of the fluorescent detectors examined demonstrated utility on each of the array platforms. For the Fc,biotin array, detector signal intensity (background adjusted) was as follows: RPE,antibody complex,>,fluospheres,>,RPE,>,liposomes,>,Alexa 546: for the IgG array: RPE/antibody complex,>,RPE,>,fluospheres,>,Alexa546,>,liposomes. The RPE,antibody complex fluoresced 67% and 150% more intensely than the next closest detector molecule for the Fc,biotin and the murine IgG arrays, respectively. A marked increase in background fluorescence (as compared to RPE alone) did not accompany the increase in signal intensity gained through RPE,antibody complex use (a true increase in signal:noise ratio). These results suggest that the RPE,antibody complex is superior to other molecules for fluorescent detection of analytes on protein microarrays. Copyright © 2002 John Wiley & Sons, Ltd. [source] Short communication: Analysis of CD4+ T-cell gene expression in allergic subjects using two different microarray platformsALLERGY, Issue 3 2008N. N. Hansel Background:, Allergic diseases are thought to involve dysregulated activation of T cells including CD4+ lymphocytes. T-cell activation results in changes in gene expression, but the optimal method to study gene expression profiles in T cells, and how this changes over time, are not known. Methods:, Circulating CD4+ T cells were obtained from subjects with atopic asthma, nonatopic asthma or nonallergic controls, and total mRNA was rapidly isolated. Atopy was defined as positive skin prick test to one of nine allergens. Gene expression was analyzed using hybridization and Affymetrix® oligonucleotide arrays (Hu133A and Hu133B chips, n = 84), or by reverse transcription-polymerase chain reaction (RT-PCR) with a pathway-targeted array (Human Th1,Th2,Th3 RT2 ProfilerTM PCR Array, Superarray, n = 16). Results:, Using Affymetrix arrays, it was difficult to discern a dominant allergy-associated profile because of heterogeneity in gene expression profiles. In contrast, a Th2-like signature was evident using RT-PCR arrays with increased expression of expected genes (e.g. IL-4, 5, 9, and 13, all P < 0.05) as well as unexpected gene transcripts (e.g. osteopontin). Gene expression profiles were relatively stable over time in circulating CD4+ T cells from two subjects using both platforms. Conclusions:, Unstimulated CD4+ T cells isolated from allergic subjects express a characteristic profile of genes when analyzed using RT-PCR based microarrays. [source] Ovarian cancer proteomics: Many technologies one goalPROTEOMICS - CLINICAL APPLICATIONS, Issue 2 2008Kothandaraman Narasimhan Abstract The last decade has seen major changes in the technologies used to identify markers for diagnosing cancer. This review focuses on recent developments on the evolving field of biomarker discovery, and validation techniques using proteomics platforms for ovarian cancer. It is possible now to diagnose various disease conditions using microliter quantities of body fluids. Currently the major developments were made in three distinct areas: (i) protein profiling, (ii) high-throughput validation techniques, and (iii) solid and liquid phase protein microarray platforms for analyzing candidate markers across subclasses and stages of cancers. The recent addition to the long list of technologies is metabolomics using metabolite profiling and informatics-based filtering of information for biomarker discovery of ovarian cancer. Emerging technologies need to address ways to eliminate the limitations posed by the complex dynamic nature of body fluids as well as ways to enrich low-abundance tumor markers if they were to become a successful biomarker discovery tool. These new technologies hold significant promise in identifying more robust markers for ovarian cancer. Since the prevalence of this disease in the population is low, the test must have a high specificity. [source] How different are luminal A and basal breast cancers?INTERNATIONAL JOURNAL OF CANCER, Issue 6 2009François Bertucci Abstract Heterogeneity of breast cancer makes its evolution difficult to predict, and its treatment far from being optimal. At least 5 main molecular subtypes exist. Two major subtypes are luminal A and basal subtypes, which have opposite features, notably survival. To characterize these 2 subtypes better, with the hope of better understanding their different biology and clinical outcome, we have profiled a series of 138 tumours (80 luminal A and 58 basal) using Affymetrix whole-genome DNA microarrays. We have identified 5,621 probe sets as differentially expressed between the 2 subtypes in our series. These differences were validated in 6 independent public series (more than 600 tumours) profiled using different DNA microarrays platforms. Analysis of functions and pathways related to these probe sets, and the extent of the observed differences, confirmed that the 2 subtypes represent very distinct entities. Genes associated with proliferation, cell cycle, cell motility, angiogenesis, and NFkB signalling were overexpressed in basal tumours. Genes involved in fatty acid metabolism, TGFB signalling, and oestrogen receptor (ER) signalling were overexpressed in luminal A samples. Half of the genes overexpressed in luminal tumours contained ER-binding sites. The number of differentially expressed genes was as high as the set of genes discriminating 2 cancers of different anatomical origin (breast and colon) or discriminating acute myeloid and lymphoid leukaemia. We provide a comprehensive list of genes/pathways that define potential diagnostic, prognostic and therapeutic targets for these 2 subtypes, which should be treated differently given the profound differences observed at the molecular level. © 2008 Wiley-Liss, Inc. 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