|
| Status |
Public on Dec 10, 2011 |
| Title |
Mouse Cumulus_8hours_GDF9-treated_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Mouse cumulus cells from cultured cumulus-oocyte complexes, growth differentiation factor 9-treated, 8 hours.
|
| Organism |
Mus musculus |
| Characteristics |
gender: female
strain: 129SV
age: 21 to 26 day old
gonadotropin priming regimen: 44 hours post equine chorionic gonadotropin treatment (eCG (5 IU))
cell type: cumulus cells
agent: GDF9
|
| Treatment protocol |
whole COCs were treated with 20 ng/ml GDF9 or 293H control medium (0.125% v/v), with or without a combination of 50 mIU/ml FSH and 10 ng/ml EGF. After 8 hours of in vitro maturation, COCs were denuded by gentle pipetting, the oocytes were removed and the cumulus cells centrifuged at 16,000g for 1 minute and snap-frozen in liquid nitrogen until further analysis.
|
| Growth protocol |
After recovery, ovaries were rinsed in Hepes-buffered tissue culture medium 199 (TCM199; MP Biomedicals, Ohio, USA) with 0.03% (w/v) polyvinyl alcohol and cilostamide (1 μM). Ovarian follicles were punctured with two syringes with 21 gauge needles and COC collected using a flame-pulled borosilicate Pasteur pipette under a 15X magnification. 20 COCs were cultured in 50 μL media drops under mineral oil overlay. COC culture media was bicarbonate-buffered α-Minimal Essential Medium with 0.03% polyvinyl alcohol, 11 mM glucose, 100 μM cysteamine, 100 IU/mL penicillin and 100 μg/mL streptomycin sulfate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction of cumulus cells was performed with the “RNeasy Mini Kit” from Qiagen (Doncaster, Victoria, Australia) according to the manufacturer’s protocol. For microarray analysis, 8 biological replicates of the experimental conditions described above were performed and RNA extracted independently. Samples were pooled in pairs to generate four replicates with enough RNA for analysis.
|
| Label |
biotin
|
| Label protocol |
The fragmented single stranded cDNA was end-labelled using terminal deoxynucleotidyl transferase enzyme and the WT Terminal Labelling kit (Millenium Sciences).
|
| |
|
| Hybridization protocol |
A total of 5ug of labelled cDNA was then hybridised to the Mouse Gene 1.0 ST Array GeneChip (Millenium Sciences) by preparing a cocktail probe (labelled cDNA at 0.025 μg/ul) that includes 1x hybridisation buffer (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01 %(v/v) Tween-20), 0.1 mg/ml herring sperm DNA, 0.5 mg/ml BSA and 7% DMSO. A total hybridisation volume of 220 μl was prepared for each sample and 200 μl loaded into a Mouse Gene 1.0 ST Array GeneChip (Affimetrix).
|
| Scan protocol |
GeneChips were washed using the appropriate fluidics script in the Affymetrix Fluidics Station 450 and then scanned using the Affymetrix GeneChip Scanner 3000.
|
| Description |
expression data from mouse cumulus cells from cultured cumulus-oocyte complexes, growth differentiation factor 9-treated, 8 hours.
|
| Data processing |
The scanner operating software, GCOS, converts the signal on the chip into a DAT file, which is then used for generating the subsequent CEL file for analysis. The initial results were imported into Partek software (St. Louis, MO, USA), and were normalized using the Robust Multichip Average (RMA) method.
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| |
|
| Submission date |
Dec 04, 2009 |
| Last update date |
Dec 10, 2011 |
| Contact name |
Maxime Sasseville |
| E-mail(s) |
[email protected]
|
| Organization name |
The Robinson Institute, University of Adelaide
|
| Street address |
Level 6, Medical school, Frome Road
|
| City |
Adelaide |
| State/province |
SA |
| ZIP/Postal code |
5005 |
| Country |
Australia |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE19327 |
Expression data from mouse cumulus cells |
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