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Ovarian Tumor Tissue Expression Study

ABSTRACT: Ovarian tumor tissues were obtained from patients with informed consent, which was approved by the Institutional Review Board of the Swedish Hospital in Seattle. Tissue acquisition, processing and storage were conducted by the Pacific Ovarian Cancer Research Consortium (POCRC) at the Fred Hutchinson Cancer Research Center. The quality of tumor tissues, the percentage of cancer cells and the histological analysis of the tumors were determined via a centralized pathology review at the POCRC repository. All samples were distributed in a de-identified manner and cannot be traced back to patients. The tumor tissues were disrupted in a Biospec Tissue-Tearor Model 985370-395 in the presence of TRIzol (Invitrogen) with a ratio of 10ml TRIzol per 50mg of tissue. Following an extraction step of 0.2 ml of Chloroform per 1ml of TRIzol, the upper aqueous phase was removed for RNA isolation. The remaining inter-phase and the phenol phase was briefly stored in 4??C for DNA isolation. The upper aqueous phase was processed for total RNA according to Invitrogen protocol and further purified using RNeasy Mini kit (Qiagen). Genomic DNA was isolated by back-extraction from the TRIzol inter-phase and the phenol phase as described on Ambion website (page 4). Detailed amplification and labeling protocols are available at the Brown lab website

ORGANISM(S): Homo sapiens

PROVIDER: GSE27223 | GEO | 2011/02/12

SECONDARY ACCESSION(S): PRJNA141967

REPOSITORIES: GEO

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Project description:All cell lines were cultured according to the specifications outlined by ATCC. Cell lines were grown to 80% confluence, then serum-deprived in 0.5% FBS for 24 hours before harvesting. Cells were washed twice with Hank's Balanced Salt Solution (Invitrogen, Carlsbad, California) and drained. 1 mL of SDS extraction buffer (0.1M NaCl, 20 mM Trizma base, 25 mM EDTA, 0.5% w/v SDS) was added directly to the plate to lyse the cells. Cellular lysate was scraped off the plates, pipetted into a cryovial, and stored at 80C until DNA extraction. Lysates were treated with 200 ug/ml ProteinaseK at 50C overnight. DNA was precipitated with one volume of Isopropanol and dissolved in TE4 buffer. Cell lines were lysed in Phenol followed by a Phenol/Chloroform extraction. The aqueous layer was added to 70% ethanol and further purified by loading the RNA/Ethanol mix into an RNeasy Mini kit (Qiagen) column. Purification was continued and finalized as described in the RNeasy kit (Qiagen) Manual. We measured transcript levels in the 27 primary ovarian tumors and 15 cell lines using the HEEBO [16] oligonucleotide microarrays, containing 44,544 70-mer probes and printed at the Stanford Functional Genomics Facility. Detailed information on the HEEBO arrays is available at the Stanford Functional Genomics Facility website. Experimental methods. Microarray experiments were performed as described at the Brown lab website. Briefly, 500 ug of total RNA from each of the ovarian tumors were amplified using Amino Allyl Message Amp II aRNA Kit (Ambion, Austin, TX, USA). The aRNAs were labeled with Cy5 and co-hybridized with Cy3 labeled Stratagene reference aRNA. For some samples, the mRNA was amplified and hybridized in duplicate or triplicate. The samples were then hybridized to HEEBO microarrays. The arrays were scanned in a low-ozone environment using a GenePix 4000A microarray scanner and images were analyzed with Genepix 5.0 (Axon instruments, Union City, CA). Using regression correlation

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Ovarian Tumor Tissue Expression Study

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