Mature MII-oocytes isolated from eCG(44-46h)/hCG (14h) treated 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). Mice were then injected with hCG (5IU/mouse), and the oviducts were removed 14 h post hCG injection. Ovulated OCCs were released by gently teasing apart the ampulla of oviducts. Denuded oocytes were obtained by incubating the mass of OCCs in medium containing 1 mg/ml of hyaluronidase for about 3-5 min at 37 ºC. Mature oocytes with a visible first polar body (hereafter termed MII oocytes) 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.
MII oocytes were washed thoroughly in the collection medium to ensure that no cumulus cells were present. Medium used for oocyte collection was the same as for collection of GV-oocytes
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
Mature MII-stage oocytes isolated from 22-24d old eCG(44-46h)/hCG(14h)- treated 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)