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Array CGH on gemcitabine resistant tumor

ABSTRACT: Biological material: One solid mouse tumor "Colon 26A" was routinely maintained by successive transplantation. A subset of mice with this tumor was treated at the maximum tolerable dosage of Gemcitabine and successively transplanted 5 times and similarly treated. The last passage was completly resistant and designated, "Colon 26G". Oligo array CGH microarray: DNA from tumor and normal liver samples were isolated using the Wizard Genomic DNA purification kit according to the manufactures protocol (Promega Benelux BV, Leiden, NL). Labeling and hybridization procedures for the oligo array CGH were performed as previously described (Carvalho et al, 2004, J. Clin. Pathol., 57:644-649) with the following modifications: a mouse oligo library version 2.0 (Compugen/Sigma-Aldrich Chemie B.V., Zwijndrecht, NL) containing 21,997 oligonucleotides (65bp) representing 21,587 exon regions on all chromosomes were spotted on the arrays. Prehybridisation was omitted and Cot-1 concentrations during the hybridization were reduced to 100 µg. Hybridizations were done using a Hybstation12 (Perkin-Elmer, Zaventem, BE). CGH arrays were scanned using a laser scanner (Agilent Technologies, Amstelveen, NL) and analyzed using Bluefuse Software v.2.0 (Bluegnome Ltd, Cambridge, UK). Images show fused values; values with confidence higher then 0.35 with the overall Cy3 and Cy5 channels normalized to a log2ratio of 0. Tumor 26G was hybridized twice and Bluefuse confidence based average values are given. No further normalizations were performed. For interpretation and visualization purposes,smoothing was performed by version 2 of aCGH smooth, with λ set to 2.0. Keywords: other

ORGANISM(S): Mus musculus

PROVIDER: GSE2373 | GEO | 2005/12/21

SECONDARY ACCESSION(S): PRJNA91527

REPOSITORIES: GEO

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Project description:This series repressents the data set from the paper "Expression microarray analysis and oligo array CGH of aquired gemcitabine resistance in mouse colon reveals selection for chromosomal aberrations". Biological material: One solid mouse tumor "Colon 26A" was routinely maintained by successive transplantation. A subset of mice with this tumor was treated at the maximum tolerable dosage of Gemcitabine and successively transplanted 5 times and similarly treated. The last passage was completly resistant and designated, "Colon 26G". Expression array: Total RNA was isolated from frozen mouse tumors using Tryzol. Single stranded cDNA was synthetized from 30 mg of total RNA by reverse transcription using aminoallyl labeled dUTP (Ambion). Labelling was performed according to the aminoallyl labeling protocol. Briefly, cDNA was incubated at room temperature for 1 h with fluorolink monofunctional cy5 or cy3 (Amersham) followed by 15 min 4 M hydroxylamine treatment. Uncoupled dyes were removed using Qiaquick PCR purification columns (Qiagen) and mixed with 12 mg poly(dA) (Amersham), 60 mg yeast tRNA (Sigma) and 24 mg Cot-1 DNA (Invitroen). The probe was dissolved in 127 ml hybridization mixture containing 46% formamide, 9.5% dextran sulfate, 2x SSC and 0.2% SDS. The probe was heated to 70Â°C for 10 min and annealed at 37Â°C for 1 h. Slides were hybridized for 14 h at 37Â°C over-night (HyArray 12â¢, Perkin Elmer). After hybridization the slides were washed in the HybArray 12â¢ with 50% formamide, 2x SSC, pH 7 at 35Â°C for 15 min, followed b PI buffer (0.1 M sodium phosphate, 0.1% Igepal Ca630, pH 8) at room temperature and 3 washes of 0.2x SSC, 0.1x SSC and 0.01x SSC at room temperature followed by centrifugation. Arrays were scanned using a laser scanner (ScanArray Express, Perkin Elmer) and analysed using ImaGene version 5.6. Cy5 cy3 ratios are calculating by taking the log2 of the signal mean of each spot minus the median background mean of all spots on the array. This is followed by a standard normalization for spot intensity and calculation of the ratios.

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Array CGH on gemcitabine resistant tumor

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