GV-stage oocytes isolated from eCG-primed (44-46h) 22-24 days old female (C57BL/6JxSJL)F1 mice
The Jackson Laboratory
Ovarian follicular development was stimulated by intraperitoneal injection of 5 IU of eCG (EMD Biosciences, Inc., Calbiochem, La Jolla, CA). Immature GV-stage oocytes were collected at 44-46 h post eCG priming by puncturing large antral follicles on ovarian surface with a pair of 26 gauge needles.
Released oocyte-cumulus cell complexes (OCCs) were collected and cumulus cells surrounding the oocyte were removed completely by passing OCCs several times through a hand-drawn small fine glass pipette with an inner diameter slightly narrower than the oocyte. Only those oocytes with an intact GV and no apparent sign of degeneration were collected. Both GV and MII oocytes were washed thoroughly in the collection medium to ensure that no cumulus cells were present. Medium used for oocyte collection was MEM-α (Invitrogen Corporation, Grand Island, NY) supplemented with 3 mg/ml of crystallized lyophilized bovine serum albumin (Sigma, St. Louis, MO), 75 mg/liter of penicillin G (Sigma) and 50 mg/liter streptomycin sulfate (Sigma). Milrinone (Sigma), a selective inhibitor of oocyte-specific phosphodiesterase (PDE3), was added into the medium at the concentration of 5 ?M to prevent the GV-stage oocytes from undergoing maturation.
Total RNA was extracted from 300 oocytes using the Picopure RNA isolation Kit (Arcturus, Mountain View, CA).
Total RNA was extracted from oocytes using the PicoPure RNA Isolation Kit according to the manufacturer?s instruction. The RNA quality and yield of each sample were determined using the Bioanalyzer 2100 and RNA 6000 Pico LabChip assay (Agilent Technologies Inc, Palo Alto, CA) in combination with Quant-iTTM RiboGreen Reagent according to supplied protocols (Invitrogen, Carlsbad, CA). Ten nanograms of total RNA from each sample was employed in the two-round cDNA synthesis and subsequent in vitro-transcription according to the Two-Cycle Eukaryotic Target Labeling Assay (Affymetrix Expression Analysis Technical Manual: ? Section 2: Eukaryotic Sample and Array Processing? (http://www.affymetrix.com/support/technical/manual/expression_manual.affx)) with the following modifications. For the first and second cycle of cDNA synthesis steps, Full Spectrum? MultiStart Primers for in vitro transcription (SystemBiosciences, Mountain View, CA) were used, instead of Affymetrix?s standard T7-Oligo(dT) primer or random hexamers.
Standard AffyMetrix procedures
Fully grown GV-stage oocytes isolated from 22-24d old eCG-primed mice.
Probe level data were imported into the R software environment and expression values were summarized using the RMA (Robust MultiChip Average) function (Irizarry et al., 2003) in the R/affy package with rma2 background adjustment, no inter-array normalization, and median polish summarization of perfect match values (Gautier et al., 2004). To account for the difference in starting RNA amount obtained from the same number of GV and MII oocytes, and based on the fact that there is no new transcriptional activity in the fully grown oocytes (De La Fuente, 2006), a normalization was performed on a sample of non-differentially expressed genes which were estimated and sampled using EM algorithm for a normal mixture model (Gautier et al., 2004; McLachlan and Krishnan, 1997)