|
| Status |
Public on Oct 14, 2012 |
| Title |
Cow_Mat_Vivo_32 |
| Sample type |
RNA |
| |
|
| Source name |
Cow follicles, mature in vivo, metaphase II, sample 32, replicate 5
|
| Organism |
Bos taurus |
| Characteristics |
tissue: ovarian follicles
Stage: mature_invivo
meiotic stage: metaphase II
development capacity: developmentally fully competent
|
| Treatment protocol |
Cumulus cells were separated from the oocytes in TCM199 culture medium (Sigma), washed in PBS and kept in TriZol reagent at -80°C prior to extraction.
|
| Growth protocol |
Holstein adult non-lactating cows 3-5 years old were used for sampling of mature COC (cumulus-oocyte complexes) after basic superovulation protocol. Adult cows of different ages and 3-6 months-old calves were used for recovery of immature COC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France). RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.35 µg RNA using the One-Color Microarray based gene Expression kit (Quick Amp Labeling, Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 µg of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 25x fragmentation buffer and 10x blocking agent following the manufacturers instructions (Agilent Technologies, Santa Clara, CA) . On completion of the fragmentation reaction, 55 µl of 2x GEx Hybridization Buffer HI-RPM (Agilent provided) was added to the fragmentation mixture and hybridized to Agilent Bovine Oligo Microarray (G2519F AMADID 015354) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2566AA) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5 µm, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in cow ovulatory follicles in which oocytes have undergone maturation (metaphase II oocytes awaiting fertilization) and are developmpentally fully competent. These ovulatory follicles were collected by ovum pick-up (OPU) in vivo
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol GE1_105_Dec08 and Grid: 015354_D_20061130) to obtain background subtracted and spatially detrended Processed Signal intensities. Poor quality features that were either saturated or non-uniform were flagged using Genespring Agilent software. Only features with a signal-to-noise ratio (SNR) of up to 2.6 and significantly different from the local background (two sided t-test) were used for further analysis. Entities were then filtered based on these flag values: at least 80% of the values in any 2 out of 3 conditions must have acceptable values. Expression values were then log2-transformed and submitted to a scale normalisation
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| |
|
| Submission date |
Mar 19, 2012 |
| Last update date |
Oct 14, 2012 |
| Contact name |
Julien Bobe |
| E-mail(s) |
Julien.Bobe@rennes.inra.fr
|
| Phone |
(33) 2 23 48 57 24
|
| Organization name |
Institut National de la Recherche Agronomique
|
| Lab |
INRA-SCRIBE
|
| Street address |
Campus de Beaulieu
|
| City |
Rennes |
| ZIP/Postal code |
35000 |
| Country |
France |
| |
|
| Platform ID |
GPL9712 |
| Series (2) |
| GSE36605 |
Transcriptional profiling of cumulus cells differentiation throughout oocyte competence acquisition in the bovine ovarian follicles. |
| GSE36617 |
Contribution of molecular actors from somatic origin to oocyte developmental competence acquisition, lessons from evolution |
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