Making and reading microarrays

Cheung, Vivian G.; Morley, Michael; Aguilar, Francisco; Massimi, Aldo; Kucherlapati, Raju; Childs, Geoffrey

doi:10.1038/4439

Cited by 572 publications

(302 citation statements)

References 12 publications

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“…A panel of 12 288 CpG island clones was created from the CGI genomic library from Cross et al, 1994, as part of the Human Genome Mapping Project Resource Center (Heisler et al, 2005). Inserts from this CGI library were polymerase chain reaction (PCR)-amplified, purified by ethanol precipitation and then arrayed onto glass microscope slides by a specialised robot designed and created at Albert Einstein College of Medicine (Cheung et al, 1999;Adrien et al, 2006). The Differential Methylation Hybridisation (DMH) technique was carried out as previously described (Yan et al, 2002).…”

Section: Cpg Island Arraysmentioning

confidence: 99%

Identification of DNA hypermethylation of SOX9 in association with bladder cancer progression using CpG microarrays

et al. 2007

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CpG island arrays represent a high-throughput epigenomic discovery platform to identify global disease-specific promoter hypermethylation candidates along bladder cancer progression. DNA obtained from 10 pairs of invasive bladder tumours were profiled vs their respective normal urothelium using differential methylation hybridisation on custom-made CpG arrays (n ¼ 12 288 clones). Promoter hypermethylation of 84 clones was simultaneously shown in at least 70% of the tumours. SOX9 was selected for further validation by bisulphite genomic sequencing and methylation-specific polymerase chain reaction in bladder cancer cells (n ¼ 11) and primary bladder tumours (n ¼ 101). Hypermethylation was observed in bladder cancer cells and associated with lack of gene expression, being restored in vitro by a demethylating agent. In primary bladder tumours, SOX9 hypermethylation was present in 56.4% of the cases. Moreover, SOX9 hypermethylation was significantly associated with tumour grade and overall survival. Thus, this high-throughput epigenomic strategy has served to identify novel hypermethylated candidates in bladder cancer. In vitro analyses supported the role of methylation in silencing SOX9 gene. The association of SOX9 hypermethylation with tumour progression and clinical outcome suggests its relevant clinical implications at stratifying patients affected with bladder cancer.

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Section: Cpg Island Arraysmentioning

confidence: 99%

Identification of DNA hypermethylation of SOX9 in association with bladder cancer progression using CpG microarrays

et al. 2007

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“…In order to remove molecular oxygen, samples were degassed via several freeze-pump-thaw cycles prior to the introduction of xenon. Figure 5; 10 Hz in Figures 4,7,8;and 20 Hz for intensities integrated in Figure 6. Ninety-degree pulses were used for acquiring spectra unless otherwise noted.…”

Section: Nmr Experimentsmentioning

confidence: 99%

“…[4][5][6][7] The spatial separation of possible interacting pairs distinguishes signals from different ligand-target combinations, and readings are made by scanning techniques or by imaging. 8 At present, particular challenges exist in the development of multiplexed assays, which allow multiple interactions in samples to be detected simultaneously.…”

Section: Introductionmentioning

confidence: 99%

Development of a Functionalized Xenon Biosensor

et al. 2004

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NMR-based biosensors that utilize laser-polarized xenon offer potential advantages beyond current sensing technologies. These advantages include the capacity to simultaneously detect multiple analytes, the applicability to in vivo spectroscopy and imaging, and the possibility of "remote" amplified detection. Here we present a detailed NMR characterization of the binding of a biotin-derivatized caged-xenon sensor to avidin. Binding of "functionalized" xenon to avidin leads to a change in the chemical shift of the encapsulated xenon in addition to a broadening of the resonance, both of which serve as NMR markers of ligand-target interaction. A control experiment in which the biotin-binding site of avidin was blocked with native biotin showed no such spectral changes, confirming that only specific binding, rather than nonspecific contact, between avidin and functionalized xenon leads to the effects on the xenon NMR spectrum. The exchange rate of xenon (between solution and cage) and the xenon spin-lattice relaxation rate were not changed significantly upon binding. We describe two methods for enhancing the signal from functionalized xenon by exploiting the laser-polarized xenon magnetization reservoir. We also show that the xenon chemical shifts are distinct for xenon encapsulated in different diastereomeric cage molecules. This demonstrates the potential for tuning the encapsulated xenon chemical shift, which is a key requirement for being able to multiplex the biosensor.

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“…For yeast experiments, DNA was labeled with allylamine-dUTP (Sigma), and reactive Cy3 and Cy5 succinamide ester monofunctional dyes (Amersham Pharmacia) were coupled to the DNA following a protocol described at www.microarrays.org. CMT-Yeast microarrays (version 1.32, Corning) containing 6135 unique ORFs were prehybridized in 35% formamide, 0.5% SDS, ‫ן4‬ SSPE, ‫ן5.2‬ Denhardt's, and 0.2 mg/mL herring sperm DNA for at least 2 h. In the meantime, ∼ 200 ng of Cy3-and Cy5-labeled yeast DNA was mixed with 15 µg of yeast tRNA (GIBCO BRL) in a final hybridization volume of 60 µL (35% formamide, 0.5% SDS, ‫ן4‬ SSPE, ‫ן5.2‬ Denhardt's; protocol adapted from Cheung et al 1999). After denaturation at 95°C for 2 min, hybridization was performed under a coverslip (LifterSlip) at 50°C for 18-19 h. Following hybridization, the coverslip was removed in ‫ן1‬ SSC, 0.1% SDS, then the microarray was washed in ‫ן2.0‬ SSC, 0.1% SDS for 10 min and in ‫ן2.0‬ SSC for 20 min ‫.…”

Section: Dna Labeling and Hybridization To Microarraysmentioning

confidence: 99%

Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array–CGH

Lage¹,

Leamon²,

Pejović³

et al. 2003

Genome Res.

233

210

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Structural genetic alterations in cancer often involve gene loss or gene amplification. With the advent of microarray approaches for the analysis of the genome, as exemplified by array–CGH (Comparative GenomicHybridization), scanning for gene-dosage alterations is limited only by issues of DNA microarray density. However, samples of interest to the pathologist often comprise small clusters of just a few hundred cells, which do not provide sufficient DNA for array–CGH analysis. We sought to develop a simple method that would permit amplification of the whole genome without the use of thermocycling or ligation of DNA adaptors, because such a method would lend itself to the automated processing of a large number of tissue samples. We describe a method that permits the isothermal amplification of genomic DNA with high fidelity and limited sequence representation bias. The method is based on strand displacement reactions that propagate by a hyperbranching mechanism, and generate hundreds, or even thousands, of copies of the genome in a few hours. Using whole genome isothermal amplification, in combination with comparative genomic hybridization on cDNA microarrays, we demonstrate the ability to detect gene losses in yeast and gene dosage imbalances in human breast tumor cell lines. Although sequence representation bias in the amplified DNA presents potential problems for CGH analysis, these problems have been overcome by using amplified DNA in both control and tester samples. Gene-dosage alterations of threefold or more can be observed with high reproducibility with as few as 1000 cells of starting material.

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Making and reading microarrays

Cited by 572 publications

References 12 publications

Identification of DNA hypermethylation of SOX9 in association with bladder cancer progression using CpG microarrays

Identification of DNA hypermethylation of SOX9 in association with bladder cancer progression using CpG microarrays

Development of a Functionalized Xenon Biosensor

Whole Genome Analysis of Genetic Alterations in Small DNA Samples Using Hyperbranched Strand Displacement Amplification and Array–CGH

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