|Public on Oct 14, 2012
|juvenile mouse follicles, immature, prophase I, replicate 2
|tissue: juvenile ovarian follicles
meiotic stage: prophase I
development capacity: developmentally incompetent
|Cumulus cells were separated from the immature oocytes by gentle pipeting. In the case of expanded cumulus of ovulated COCs, they were transferred into M2-droplets containing 300 µg/ml of hyaluronidase (Sigma-Aldrich) for a few seconds to disperse granulosa cells. In all cases, media containing granulosa cells after oocyte denudation and elimination were centrifuged and cumulus cell pellets rinsed in PBS before snap-freezing in liquid nitrogen and storage at -70° until RNA extraction.
|Immature, unexpanded cumulus-oocytes complexes (COC) were isolated from prepubertal (3-week-old) or adult (8-week-old) B6CBAF1/J mice ovaries by puncturing large follicles out of the ovaries in M2 culture medium (Sigma). Mature, expanded COCs were obtained from the oviduct from 8-week-old mice after standard superovulation 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).
|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.
|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 (G4846A) 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).
|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%).
|Gene expression in juvenile mouse follicles in which prophase I oocytes are meiotically competent but developmentally incompetent after meiosis resumption
|The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid:026655_D_F_20100123) 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
|Mar 19, 2012
|Last update date
|Oct 14, 2012
|(33) 2 23 48 57 24
|Institut National de la Recherche Agronomique
|Campus de Beaulieu
|Transcriptional profiling of cumulus cells differentiation throughout oocyte competence acquisition in the murine ovarian follicles.
|Contribution of molecular actors from somatic origin to oocyte developmental competence acquisition, lessons from evolution