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Series GSE29763 Query DataSets for GSE29763
Status Public on Jun 09, 2016
Title Gene expression profile associated with a cytostatic does of mifepristone in ovarian cancer cells by microarray analysis
Organism Homo sapiens
Experiment type Expression profiling by array
Summary Goal of the experiment: To examine differential gene expression in three ovarian cancer cell lines in the absence and presence of mifepristone.

Brief description of the experiment:
Hypothesis: The gene expression profile associated with a cytostatic response to Mifepristone will be similar between ovarian cancer cells regardless of their p53 status or sensitivity to Cisplatin.
Aim: Evaluate the modifications in gene expression profile in ovarian cancer cells in response to a cytostatic concentration of Mifepristone against those in logarithmic phase of growth (Vehicle). Compare the gene expression profile of cells with different genetic backgrounds as well as between those with identical genetic backgrounds but different sensitivities to Cisplatin.
Cell Types: SKOV-3 as null p53 and semi-resistant to Cisplatin, OV2008 cells as wild type p53 and hyper-sensitive to Cisplatin , OV2008/C13 cells as wild type p53 and resistant to Cisplatin.
Conditions: Cells from three different passages were cultured in T25 flasks. The starting number of cells was 250,000 or 500,000, however cells were left to grow for 48 hr or 24 hr respectively before to start the treatment with Mifepristone 8.6 µg/ml (20 µM) or DMSO (vehicle) for 24 hr.
 
Overall design Experimental design: Three cell lines of ovarian cancer cells were grown in culture: SK-OV-3, OV2008, and OV2008/C13. Three different passages of cells of each cell line were used for each treatment (vehicle control and mifepristone) (n=3). Total RNA was extracted from ovarian cancer cells and processed for hybridization to Codelink Whole Human Genome microarrays.

Quality control steps: The cRNA that was synthesized from each passage of each cell line was used for hybridization to a single CodeLink (Applied Microarrays, Tempe, AZ) whole human genome microarray. Only one sample was hybridized with each slide and only one dye (Alexa 647) was used so no dye swaps were necessary. Bacterial control spikes were used as per manufacturer's instructions.

Samples used, extract preparation and labeling:
The origin of each biological sample: The samples were ovarian cancer cell lines.

Manipulations of biological samples and protocols used: Cells were in log phase of growth (60-70% confluent) when treatment was initiated. Cells were treated with vehicle (DMSO) or with 20 micromolar mifepristone for 24 hours.

Experimental factor: treatment with vehicle or with 20 micromolar mifepristone for 24 hours

Technical protocols: One ml TRI reagent was added to the cultured ovarian cancer cells. Bromochloropropane and sodium acetate were added and the samples were centrifuged to separate the phases. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen) (Eyster and Brannian, Meth Molec Biol 2010;590:71-89). The purity and quantity of RNA were evaluated by spectrophotometry and agarose gel electrophoresis.

Labeled cRNA was prepared using the MessageAmp II-Biotin enhanced kit (Ambion). 1.0 microgram of total ovarian cancer cell RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on QIAquick columns (Qiagen) and the eluent was dried down in a SpeedVac concentrator. The double-stranded cDNA was resuspended in a mixture containing T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on RNA extraction columns. The concentration of cRNA was determined by spectrophotometry.

Hybridization procedures and parameters: 10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the CodeLink Whole Human Genome microarray slides, and hybridized for 18 hours at 37C.
The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The Alexa fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and twice in 0.05% Tween 20 for 5 sec each. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.

Keywords: ovarian cancer, cell lines, mifepristone, RU486
 
Contributor(s) Eyster KM, Hapon MB, Gamarra Luques CD, Callegari EA, Goyeneche AA, Telleria CM
Citation(s) 27233943
Submission date Jun 06, 2011
Last update date Sep 08, 2016
Contact name Kathleen M Eyster
E-mail(s) Kathleen.Eyster@usd.edu
Organization name University of South Dakota
Department Basic Biomedical Sciences
Street address 414 E. Clark St.
City Vermillion
State/province SD
ZIP/Postal code 57069
Country USA
 
Platforms (1)
GPL2895 GE Healthcare/Amersham Biosciences CodeLink Human Whole Genome Bioarray
Samples (18)
GSM737588 ovarian cancer cell line SKOV-3 rep 1
GSM737589 ovarian cancer cell line SKOV-3 rep 2
GSM737590 ovarian cancer cell line SKOV-3 rep 3
Relations
BioProject PRJNA141027

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE29763_RAW.tar 131.1 Mb (http)(custom) TAR (of TXT)
Processed data included within Sample table
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