|
| Status |
Public on Apr 25, 2011 |
| Title |
Mature oocyte in vitro rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Mature in vitro oocyte
|
| Organism |
Bos taurus |
| Characteristics |
cell type: matured oocytes
|
| Treatment protocol |
Immature cumulus oocyte complexes (COCs) were obtained by aspirating 3 to 8 mm follicles on the ovaries collected from cows slaughtered at a local abattoir. Good quality COCs, judged morphologically with multiple cumulus layers and homogenous cytoplasm, were selected under the stereo microscope and washed repeatedly in phosphate-buffered saline (PBS). Following washing, half of the COCs were immediately denuded by repeated pippeting in PBS. Intact oocytes with no cumulus traces were pooled in groups of 10 oocytes per tube and immediately snap frozen in liquid nitrogen. The remaining intact COCs were incubated in maturation medium [TCM-199 supplemented with 10% (v/v) fetal calf serum and 10 ng/ml epidermal growth factor] in 4-well dishes (Nunc, Roskilde, Denmark) at 39°C for 24 h under an atmosphere of 5% CO2 in air with maximum humidity. At 24 h, in vitro matured (IVM) COCs were denuded and snap frozen as described above.
|
| Growth protocol |
Immature bovine oocytes were collected immediately (T0) while in vitro matured oocytes were collected after 24 h of submission to maturation medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction was carried out using PicoPure RNA Isolation kit (Arcturus Bioscience, Mountain View, CA, USA), by incorporating a DNase treatment step using RNase-free DNase set (Qiagen, West Sussex, UK), according to the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
100 ng of total RNA was subjected to two rounds of linear amplification using the GeneChip® Expression 3’-Amplification Two-Cycle cDNA Synthesis kit (Affymetrix Inc., Santa Clara, CA) according to the manufacturer’s instructions. cDNA was synthesized during the first cycle and biotin-labeled nucleotides were incorporated during the second in vitro transcription reaction.
|
| |
|
| Hybridization protocol |
Following fragmentation, 15 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Bovine Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
GeneChips were scanned using the GeneChip® Scanner 3000 (Affymetrix).
|
| Description |
Gene expression from mature in vitro derived bovine oocyte
|
| Data processing |
The data was analysed using the R library farms (Factor Analysis for Robust Microarray Summarization).
|
| |
|
| Submission date |
Aug 06, 2010 |
| Last update date |
Apr 25, 2011 |
| Contact name |
Paul McGettigan |
| E-mail(s) |
pmcget02@gmail.com
|
| Organization name |
University College Dublin
|
| Department |
School of Agriculture, Food Science and Veterinary Medicine
|
| Lab |
Animal Genomics Laboratory
|
| Street address |
Veterinary Sciences Centre, UCD
|
| City |
Belfield |
| State/province |
Co Dublin |
| ZIP/Postal code |
Dublin 4 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL2112 |
| Series (1) |
| GSE23449 |
Gene Expression Profiles in Immature and In vitro matured bovine Oocytes |
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