Expression profiling using a hexamer-based universal microarray

Roth, Matthew E.; Li, Feng; McConnell, Kevin J.; Schaffer, Paul J.; Guerra, Cesar E.; Affourtit, Jason P.; Piper, Kevin R.; Guccione, Lorri; Hariharan, Jayashree; Ford, M. J.; Powell, Stephen W; Krishnaswamy, Harish; Lane, J. G.; Guccione, Lisa; Intrieri, Gino; Merkel, Jane S.; Perbost, Clotilde; Valerio, Anthony; Zolla, Brenda; Graham, Carol D; Hnath, Jonathan; Michaelson, Chris; Wang, Rixin; Ying, Baoge; Halling, Conrad; Parman, Craig E; Raha, Debasish; Orr, Brent A.; Jedrzkiewicz, Barbara; Liao, Ji; Tevelev, Anton; Mattessich, Martin J; Kranz, David M.; Lacey, Michelle; Kaufman, Joseph C; Kim, Junhyong; Latimer, Darin R; Lizardi, Paul M.

doi:10.1038/nbt948

Cited by 32 publications

(26 citation statements)

References 43 publications

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“…Microarray analysis was performed with the GenCompass universal microarray system (Agilix Corporation, New Haven, Conn.). The specific details of this technology have been described recently (33). The data reference the UniGene database Build 161 (National Center for Biotechnology Information, Bethesda, Md.).…”

Section: Methodsmentioning

confidence: 99%

“…)-or ethidium bromide (Sigma)-stained gels. For quantitative real-time PCRs (qPCRs), primers and probes were designed with Beacon Designer 2.06 (Premier Biosoft International, Palo Alto, Calif.), and analyses were performed using the MX400 Multiplex Quantitative PCR system (Stratagene, La Jolla, Calif.) as previously described (33). Primers and probes for all analyses are available upon request.…”

Section: Methodsmentioning

confidence: 99%

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Down-Regulation of Rad51 and Decreased Homologous Recombination in Hypoxic Cancer Cells

Bindra¹,

Schaffer²,

Meng

et al. 2004

Molecular and Cellular Biology

335

291

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There is an emerging concept that acquired genetic instability in cancer cells can arise from the dysregulation of critical DNA repair pathways due to cell stresses such as inflammation and hypoxia. Here we report that hypoxia specifically down-regulates the expression of RAD51, a key mediator of homologous recombination in mammalian cells. Decreased levels of Rad51 were observed in multiple cancer cell types during hypoxic exposure and were not associated with the cell cycle profile or with expression of hypoxia-inducible factor. Analyses of RAD51 gene promoter activity, as well as mRNA and protein stability, indicate that the hypoxiamediated regulation of this gene occurs via transcriptional repression. Decreased expression of Rad51 was also observed to persist in posthypoxic cells for as long as 48 h following reoxygenation. Correspondingly, we found reduced levels of homologous recombination in both hypoxic and posthypoxic cells, suggesting that the hypoxia-associated reduction in Rad51 expression has functional consequences for DNA repair. In addition, hypoxia-mediated down-regulation of Rad51 was confirmed in vivo via immunofluorescent image analysis of experimental tumors in mice. Based on these findings, we propose a novel mechanism of genetic instability in the tumor microenvironment mediated by hypoxia-induced suppression of the homologous recombination pathway in cancer cells. The aberrant regulation of Rad51 expression may also create heterogeneity in the DNA damage response among cells within tumors, with implications for the response to cancer therapies.Solid tumors constitute a unique tissue type, characterized by hypoxia, low pH, and nutrient deprivation (45). Although decreased oxygen tension is potentially toxic to normal human cells, cancer cells acquire genetic and adaptive changes allowing them to survive and proliferate in a hypoxic microenvironment. Intratumoral hypoxia induces profound alterations in numerous physiological processes, including altered glucose metabolism, up-regulated angiogenesis, increased invasive capacity, and dysregulation of apoptotic programs (37).From a clinical standpoint, many studies have established hypoxia as an independent and adverse prognostic variable in patients with head and neck, cervical, or soft tissue (sarcoma) tumors (3,26). With regard to the extent of hypoxia observed in tumors, it has been proposed that cells within hypoxic regions of solid tumors often derive almost all metabolic energy requirements from up-regulated glycolytic pathways. This phenomenon has been referred to as the Pasteur effect (34) and provides a partial physiologic explanation for the viability of tumor cells exposed to severe hypoxia within the tumor microenvironment. Polarographic needle electrode studies used to measure oxygen tension directly in cancer patients have revealed that a significant proportion of breast carcinomas (up to 40%) contain regions of severely decreased oxygen tension (0 to 2.5 mm Hg, compared to the normal tissue range of 24 to 66 mm Hg) while still ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Down-Regulation of Rad51 and Decreased Homologous Recombination in Hypoxic Cancer Cells

Bindra¹,

Schaffer²,

Meng

et al. 2004

Molecular and Cellular Biology

335

291

View full text Add to dashboard Cite

show abstract

“…The 5 end-phosphorylated 33-mer is designed to fold into itself to form a 10-bp stem, a 4-base loop, and a single-stranded 9-base 3 extension containing a degenerate hexamer region with Cy5 blocking the terminal 3 end. These partial single-stranded target mixtures mimic the annealed targets for ligation to hexamer microarrays in the universal microarray system (UMAS) (9). …”

Section: Methodsmentioning

confidence: 99%

“…The sum of all pixel intensities were captured per spot, and averages were calculated from triplicate spots in the array. For a test of microarray probe functionality, ligation reactions were carried out as previously described (9). A 33-mer Cy5-labeled degenerate hairpin oligonucleotide (Integrated DNA Technologies) was used as a surrogate target mixture.…”

Section: Methodsmentioning

confidence: 99%

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Analysis of oligonucleotide microarrays by 3′ end labeling using fluorescent nucleotides and terminal transferase

Guerra¹

2006

BioTechniques

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A simple enzymatic labeling procedure is described to determine spot quality in oligonucleotide microarrays. By using fluorescently labeled dideoxynucleotides or ribonucleotides as substrate for terminal deoxynucleotidyl transferase (TdT), a single fluorophore can be covalently attached at the 3' end of each oligonucleotide probe molecule in the spot. Fluorescein-12-ddUTP CyTM3-ddUTP Cy5-UTP, and Cy3-UTP were compared as TdT substrates for 3' end labeling an array of 1273 hexamer probes. Cy5-UTP was found to show minimal bias toward probe base composition and is therefore well suited for quantitative analysis of microarray spots where the oligonucleotide probes are coupled via a 5' end linkage to the solid phase.

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Sensory ability in the narwhal tooth organ system

Nweeia

Eichmiller

Hauschka

et al. 2014

The Anatomical Record

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The erupted tusk of the narwhal exhibits sensory ability. The hypothesized sensory pathway begins with ocean water entering through cementum channels to a network of patent dentinal tubules extending from the dentinocementum junction to the inner pulpal wall. Circumpulpal sensory structures then signal pulpal nerves terminating near the base of the tusk. The maxillary division of the fifth cranial nerve then transmits this sensory information to the brain. This sensory pathway was first described in published results of patent dentinal tubules, and evidence from dissection of tusk nerve connection via the maxillary division of the fifth cranial nerve to the brain. New evidence presented here indicates that the patent dentinal tubules communicate with open channels through a porous cementum from the ocean environment. The ability of pulpal tissue to react to external stimuli is supported by immunohistochemical detection of neuronal markers in the pulp and gene expression of pulpal sensory nerve tissue. Final confirmation of sensory ability is demonstrated by significant changes in heart rate when alternating solutions of high‐salt and fresh water are exposed to the external tusk surface. Additional supporting information for function includes new observations of dentinal tubule networks evident in unerupted tusks, female erupted tusks, and vestigial teeth. New findings of sexual foraging divergence documented by stable isotope and fatty acid results add to the discussion of the functional significance of the narwhal tusk. The combined evidence suggests multiple tusk functions may have driven the tooth organ system's evolutionary development and persistence. Anat Rec, 297:599–617, 2014. © 2014 Wiley Periodicals, Inc.

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Expression profiling using a hexamer-based universal microarray

Cited by 32 publications

References 43 publications

Down-Regulation of Rad51 and Decreased Homologous Recombination in Hypoxic Cancer Cells

Down-Regulation of Rad51 and Decreased Homologous Recombination in Hypoxic Cancer Cells

Analysis of oligonucleotide microarrays by 3′ end labeling using fluorescent nucleotides and terminal transferase

Sensory ability in the narwhal tooth organ system

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