SKOV-3TR
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE2627 | GEO | 2007/10/31
SECONDARY ACCESSION(S): PRJNA92141
REPOSITORIES: GEO
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Project description:Human paclitaxel-resistant (SKOV-3TR) ovarian cancer cell line and the parent SKOV-3 cell line were grown in RPMI medium containing 10% fetal calf serum. The experiment was done to compare the expression differences between the parent SKOV-3 cell line and the derived SKOV-3TR cell line. Cells were plated in T75 flasks. They were cultured until they were 80% confluent and harvested by trypsinization followed by pelleting of the cells. All RNA isolation was accomplished with the Tri-Reagent (SIgma) and purified using the Qiagen RNeasy Mini Kit reagents. The RNA (50 ug) was annealed with a random hexamer primer, and reverse-transcribed into cDNA with Powerscript (Clontech) reverse transcriptase for 2h at 42 degrees in the presence of amino-allyl dUTP. The cDNA from SKOV-3 RNA and SKOV-3TR RNA was covalently coupled separately with Cy3 and Cy5 monoreactive fluors in 50 mM sodium bicarbonate, pH 9.0. The Cy3 and Cy5 labelled cDNAs were combined and purified with a QIAquick PCR purification kit and hybridized to the microarray using the Hybrite chamber (Vysis). Fluorescent array images were collected for both Cy3 and Cy5 with a ScanArray 4000 (Perkin Elmer) at a resolution of 10 um at maximum laser power and photo-multiplier tube voltage of 50 to 60%. Segmentation was performed using the histogram method of the QuantArray (Perkin Elmer) package. Data was transformed into log base 2 expression ratios and normalized using scaled loess normalization.
2010-05-25 | E-GEOD-2627 | biostudies-arrayexpress
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Project description:We used oligonucleotide microarrays to find differentially expressed genes between control subjects and those affected by sporadic amyotrophic lateral sclerosis. Experiment Overall Design: Complementary RNAs (cRNAs) labelled with Cy5-CTP (Perkin-Elmer) were synthesized from 1 µg of total RNA of each sample of human motor cortex using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies) following the manufacturerâs protocol. A reference cRNA, labelled with Cy3-CTP (Perkin-Elmer), was synthesized from 1 µg of sample Diseased 2 RNA. Aliquots (750 ng) of Cy3 and Cy5 labelled cRNA targets were co-hybridized on Whole Human Genome Oligo Microarrays (Agilent Technologies). Microarray hybridization and washing were performed using reagents and instruments (hybridization chambers and rotating oven) as indicated by the manufacturer (Agilent Technologies). Microarrays were scanned at 10-μm resolution using a GenePix Personal 4100A microarray scanner and the GenePix Pro 6.0 acquisition and data-extraction software (Molecular Devices, Corp.).
2008-06-13 | E-GEOD-4595 | biostudies-arrayexpress
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 70°C for 10 min and annealed at 37°C for 1 h. Slides were hybridized for 14 h at 37°C over-night (HyArray 12â¢, Perkin Elmer). After hybridization the slides were washed in the HybArray 12⢠with 50% formamide, 2x SSC, pH 7 at 35°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.
2010-06-05 | E-GEOD-2352 | biostudies-arrayexpress
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2015-12-09 | E-GEOD-75783 | biostudies-arrayexpress
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2006-01-05 | GSE3972 | GEO
Project description:Gene expression of adherent cells in a coculture of the human bone marrow stromal cell line HS-27a and peripheral blood monocytes (CD14+ cells) was compared to HS-27a cultured alone. Microarray experiments were conducted using CD14+ cells from 7 healthy donors over a period of >7 months. Monocytes were isolated by ficoll separation of whole blood, followed by labelling cells with CD14 monoclonal antibody Tuek4 and magnetic bead selection (Milteny rat anti mouse IgG2a+2b beads) on an AutoMacs. HS27a cells were plated in T75 flasks at approximately 80% confluence. The next day 1.5x10E6 freshly isloated human CD14+ cells were added to the culture. At 3 days the cultures were washed 3x with Hank’s buffer to remove nonadherent monocytes. Co-cultures and HS27a cultured alone were harvested by brief trypsinization followed by pelleting of the cells. All RNA isolation was accomplished with Qiagen RNeasy Mini Kit reagents. The RNA (25 ug) was annealed with 5 ug oligo dT12-18, and reverse-transcribed into cDNA with Superscript II reverse transcriptase for 2h at 42ºC in the presence of 0.5mM dGTP, 0.5mM dCTP, 0.5mM dATP, 0.3mM dTTP, 0.2mM amino-allyl dUTP. After hydrolysis of RNA in 0.2M NaOH, Tris was removed from the reaction with a Microcon-30 concentrator. The cDNA from HS27a and HS27a-monocyte cocultures was covalently coupled separately with Cy5 and Cy3 monoreactive fluors, respectively, in 50mM sodium bicarbonate, pH 9.0, followed by quenching with 2.7M hydroxylamine. The Cy5 and Cy3 labelled cDNAs were combined and purified with a QIAquick PCR purification kit and suspended in 36 ul of 3X SSC and 0.8 mg/ml of poly-A for hybridization to the microarray. Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000A fluorescent scanner and image intensity data were extracted and analyzed with GenePix Pro 3.0 analysis software. After background correction and removal of flagged values, log base 2 expression ratios were mean centered and linear transformed to obtain the log and linear values given in the data tables. Keywords: repeat sample
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