|
| Status |
Public on Oct 14, 2012 |
| Title |
Xen_StageIV_13 |
| Sample type |
RNA |
| |
|
| Source name |
xenopus follicles, stage IV, immature, prophase I, replicate 2
|
| Organism |
Xenopus laevis |
| Characteristics |
tissue: stage IV ovarian follicles
Stage: immature
meiotic stage: prophase I
development capacity: developmentally incompetent
|
| Treatment protocol |
The monolayers of follicular cells (=granulosa cells) surrounding the oocytes were manually recovered after a 30-min treatment with collagenase in calcium-free OR2 buffer and washed in PBS. Cell pellets were snap-frozen in liquid nitrogen after addition of TRIzol reagent and stored at -70°C until RNA extraction.
|
| Growth protocol |
Sexually mature adult Xenopus laevis females (>3 year-old) were used for sampling immature vitellogenic (stage IV) and post-vitellogenic (stage VI) ovarian follicles. Mature follicles were obtained by incubating immature post-vitellogenic follicles for 15 h at room temperature in OR2 buffer (83 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na2HPO4, 5 mM HEPES, pH 7.4) supplemented with 40 IU/mL of human chorionic gonadotropin.
|
| 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 0.35µg 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 015066) 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 stade IV xenopus follicles in which prophase I oocytes are meiotically competent but developmentally incompetent after meiosis resumption
|
| 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: 015066_D_20060913) 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 |
GPL11258 |
| Series (2) |
| GSE36603 |
Transcriptional profiling of somatic cells differentiation throughout oocyte competence acquisition in the xenopus ovarian follicles. |
| GSE36617 |
Contribution of molecular actors from somatic origin to oocyte developmental competence acquisition, lessons from evolution |
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