title: Dexamethasone-regulated genes in M-1 CCD cells
status: Public on Aug 26 2005
contact.city: Baltimore
contact.first: Djikolngar
contact.last: Maouyo
contact.email: dmaou001@umaryland.edu
contact.state: MD
contact.country: USA
contact.institute: University of Maryland Baltimore
contact.phone: 410-706-2421
organism: Mus musculus
description: Experimental design. Cells were then treated with 100 nM hydrosoluble dexamethasone or steroid-free medium for 3 hours at 37˚C with 5% CO2. RNA samples from dexamethasone-treated and control cells were extracted with Trizol reagent, and DNA-decontaminated using DNA-free kit, commercially available (Ambion, Austin, TX). Yield and concentration was quantified by spectrophotometry. RNA quantity and integrity was confirmed by denaturing formaldehyde agarose gel electrophoresis and ethidium bromide staining. These samples were used for microarray labeling. Microarrays, RNA labeling, and hybridization. High quality spotted cDNA on glass slide microarrays were purchased from the Microarray Centre of the University Health Network (Toronto, Canada). These arrays contained PCR-generated cDNA inserts of all 15,264 sequence-verified mouse ESTs, from the National Institute of Aging collection. Duplicate 100-mm diameter cDNA spots are represented on GAPS II slides (Corning, Inc.). Spot cDNA identity is tracked by composed identifiers including clone identifier, description, and Unigene, and catalogued as “M15Kv1”. Briefly, 5 g total RNA was reverse-transcribed using oligo-dT flanked at 5’ end with capture sequences specific to fluorescent Cy3- or Cy5-coupled dendrimer complexes. The resulting cDNAs from the control and test samples were mixed and hybridized with the mouse cDNA arrays in humidified hybridization chambers at 60˚C for 18 hours. Following hybridization, microarrays were washed three times as described above. Quantitation. Slides were read using a laser confocal scanner, the GSI Lumonics ScanArray 5000 imaging system, at 10-µm resolution set up for excitation/emission at 543/570 and 649/670 nm, and for acquisition of Cy3 and Cy5-labeled spots respectively. Scanned image data were saved in 16-bit tiff files and quantitated with Imagene 4.0 (BioDiscovery, Marina del Rey, CA). The mean intensities of fluorescent spots were measured.
manufacture_protocol: High quality spotted cDNA on glass slide microarrays were purchased from the Microarray Centre of the University Health Network (Toronto, Canada). These arrays contained PCR-generated cDNA inserts of all 15,264 sequence-verified mouse ESTs, from the National Institute of Aging collection. Duplicate 100-mm diameter cDNA spots are represented on GAPS II slides (Corning, Inc.).