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Microarray-based CGH using BAC clones

ABSTRACT: High resolution BAC array used to study changes in the copy number of chromosome 21 in Acute Meyloid Leukemia patients Genomic DNA from patients as well as from normal donors were differentially labelled and hybridized on the array 36 RPCI-11 BAC clones covering the q-arm of chromosome21 from position15.1MB(close to the centromere)to the telomeric position 46.9 MB with an average gap between clones of 800 kb (range 346-1593 kb)In addition 23 randomly selected clones, each representing 1 of the remaining chromosomes1-20,22,X and YPositions of genes and BAC clones were determined according to the NCBI (http://www.ncbi.nlm.nih.gov/mapview) 59 BAC clones spotted in 6 replicas for a total 354 spots on GAPII corning glass slides using Affymetrix GMS417 Affymetrix Arrayer with 4-ring&pin configuration. (Affymetrix,Santa Clara, CA). Purified clones spotted in 50%DMSO DNA Isolation of these clones was performed from bacterial culture (250ml) using Qiagen midi kit (Qiagen, Valencia CA) gDNA from AML patients and healthy controls was extracted and labelleb according to the published protocol by JR Pollack in Nature Genetics vol.23 Sep.99, also available http://cmgm.stanford.edu/pbrown/protocols/4_genomic.html Fluorescence intensity ratios were measured in control experiments (reference versus reference hybridization) to weight DNA Amplification or deletion Fluorescence ratio >1 indicates amplification, Fluorescence ratio <1 indicate deletion Mean of the background corrected pixel-by-pixel fluorescence ratio between the two dyes at each spot was obtained by genePix software. Series_platform_id: my_array13 Series_submitter weblink: www.dnaarrays.org Keywords: other

ORGANISM(S): Homo sapiens

PROVIDER: GSE1078 | GEO | 2004/02/29

SECONDARY ACCESSION(S): PRJNA87177

REPOSITORIES: GEO

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Project description:Experiment on the effects of ethanol (acute oral) on induction of genes by polyinosinic polycytidylic acid (poly IC). Microarray methodology follows: cDNA for each sample was synthesized from 8.5 µg total RNA using a Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) in combination with a T7-(dT)24 primer. Following phenol-chloroform extraction of the cDNA, biotinylated cRNA was transcribed in vitro using the BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY) and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA). Twenty micrograms of purified cRNA was fragmented by incubation in fragmentation buffer (200 mM Tris-acetate, pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate) at 94°C for 35 minutes and chilled on ice. Fifteen micrograms of fragmented biotin-labeled cRNA was hybridized to the Murine Genome U74Av2 Array (Affymetrix, Santa Clara, CA), interrogating a total of 12,488 murine gene cDNA probes, for 16 hr at 45°C with constant rotation (60 rpm). The arrays were washed and then stained for 10 min at 25°C with 10 µg/mL streptavidin-R phycoerythrin (Vector Laboratories, Burlingame, CA) followed by 3 µg/mL biotinylated goat anti-streptavidin antibody (Vector Laboratories) for 10 minutes at 25°C. Arrays were then stained once again with streptavidin-R phycoerythrin for 10 min at 25°C. After washing and staining, the arrays were scanned using an Agilent GeneArray Scanner (Agilent Technologies) at 570 nm. Pixel intensities were measured, expression signals were analyzed and features extracted using a commercial software package (Microarray Suite ver 5.0, Affymetrix). Data mining and statistical analyses were performed with Data Mining Tool ver 3.0 (Affymetrix) algorithms. Arrays were globally scaled to a target intensity value of 2,500 in order to compare individual experiments. The absolute call (present, marginal, absent) of 12,488 gene expressions in each sample, as well as the direction of change, and fold change of gene expressions between samples were identified using the above-mentioned software. Keywords: other

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Microarray-based CGH using BAC clones

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