|
| Status |
Public on Dec 31, 2014 |
| Title |
Ovary,Mouse190,tumor |
| Sample type |
RNA |
| |
|
| Source name |
tumor
|
| Organism |
Mus musculus |
| Characteristics |
strain: outbred
mouse id: mouse 190
genotype/variation: Arid1afl/fl;(Gt)Rosa26PIK3CA*H1047R
injected with: AdCRE virus particles (the right ovary)
tissue type: Ovarian clear cell carcinoma
|
| Treatment protocol |
To induce genetic recombination in the ovarian surface epithelium (OSE), we employed a modified version of the ex-vivo AdCRE intrabursal delivery method. Briefly, AdCRE particles (obtained from the University of Iowa Gene Transfer Core) were diluted in sterile 1X Dulbeccos’s phosphate buffered saline (dPBS) containing 8 μg/mL polybrene. Deeply anesthetized 8-10 week old mice were given a single 5 μL injection of AdCRE particles (2.5E7 plaque-forming units or pfu) into the right ovarian bursal cavity of the surgically exposed ovary using a sterile 31 gauge needle. Immediately following AdCRE injection, the surgically excised ovaries were washed thoroughly with sterile 1X dPBS and placed back into the abdominal cavity. The surgeries were performed on multiple occasions. Age- and genotype-matched, non-littermate animals were randomized prior to AdCRE injection. The surgeon was blinded to the genotypes prior to surgery and AdCRE injection. All surgical procedures were performed in accordance with protocols approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee.
|
| Growth protocol |
All mice were maintained at the University of North Carolina at Chapel Hill, Animal Facility using standard techniques in accordance with protocols approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from pulverized tumor samples or ovarian epithelial cells using the TRIzol method (Invitrogen), followed by an RNA cleanup step and on-column DNA digestion using the RNAeasy mini prep kit (Qiagen) according to the manufacturers’ instructions. The WT Expression HT Kit for Robotics (Ambion) was used to generate sense-strand cDNA from total RNA.
|
| Label |
biotin
|
| Label protocol |
Following synthesis of sense-strand cDNA, the cDNA was fragmented and labeled with the Affymetrix GeneChip HT Terminal Labeling Kit.
|
| |
|
| Hybridization protocol |
The Beckman Coulter Biomek FXP Laboratory Automation Workstation with the Target Express set up was used to prepare the samples with these two kits. Fragmented and labeled cDNA was used to prepare a hybridization cocktail with the Affymetrix GeneTitan Hybridization Wash and Stain Kit for WT Arrays.
|
| Scan protocol |
Hybridization, washing, staining and scanning of the Affymetrix Mouse Gene 2.1 ST peg plate arrays was carried out using the Affymetrix GeneTitan MC Instrument. GeneChip Command Console Software (AGCC) was used for GeneTitan Instrument control. Affymetrix Expression Console Software was used for basic data analysis and quality control.
|
| Description |
OV190 1T
|
| Data processing |
Affymetrix CEL files were normalized using the Robust Multichip Average normalization method. Expression changes (tumor versus control) were determined using Linear Models for Microarray Data Analysis (LIMMA) or Significance Analysis of Microarray (SAM) analysis. Probes with a false discovery rate (FDR) of 0% were considered statistically significant.
|
| |
|
| Submission date |
May 07, 2014 |
| Last update date |
Dec 31, 2014 |
| Contact name |
Jonathan C Schisler |
| E-mail(s) |
schisler@unc.edu
|
| Phone |
919-843-8708
|
| Organization name |
The University of North Carolina at Chapel Hill
|
| Department |
McAllister Heart Institute
|
| Lab |
Schisler Lab
|
| Street address |
MBRB, Rm 2340C
|
| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599-7126 |
| Country |
USA |
| |
|
| Platform ID |
GPL17400 |
| Series (1) |
| GSE57380 |
Coexistent ARID1A-PIK3CA mutations promote ovarian clear cell tumorigenesis through pro-tumorigenic inflammatory cytokine signaling |
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