As described in growth protocol, no other treatment applied.
Growth protocol
Ovaries were washed and 2-6 mm follicles were aspirated using an 18gauge needle. Only oocytes surrounded by several cell layers of dense cumulus were selected for culture. Oocytes were washed three times in TL-HEPES (0.22mM sodium pyruvate, 3mg/ml BSA, pH 7.4) and placed 10 per 50 ml drop of maturation media that had been equilibrated in 5% carbon dioxide and air (39°C, high humidity). Maturation medium consisted of TC199 supplemented with 3ml of bovine LH and FSH (Sioux Biochemical), 0.25mM sodium pyruvate, and 10% fetal calf serum. After 20 hours of incubation in maturation media, oocytes were removed, washed in TL-HEPES and placed in groups of 10 oocytes each into 44 ul microdrops of IVF-Talp (Biowhittaker, Walkersburg, MD) supplemented with 0.22mM sodium pyruvate, and 6 mg/ml essentially fatty acid free BSA. Live sperm were separated by centrifugation in a percoll gradient (Sigma, P1985). Eggs were fertilized by adding one million/ ml of sperm, 2.0ug heparin (Sigma H-3125), and pencillamine, hypotaurine, epinephrine (Sigma-P-4875, H-1384, E-4250) to each drop of fertilization media. Oocytes were removed from fertilization media after 18-22 hours, washed in TL-HEPES, placed in a 1.5 ml tube, and vortexed for 2 min to remove cumulus cells. Twenty-five cumulus free oocytes were placed in each 50ul drop of SOF culture medium, supplemented with 8 mg/ml of fatty acid free BSA, nonessential and essential amino acids, and pyruvate.
Extracted molecule
total RNA
Extraction protocol
Total RNA extraction was carried out using the mirVana RNA extraction kit according to the manufacturers protocols (Applied Biosciences/Ambion, Austin, TX ).
Label
biotin
Label protocol
RNA was prepared utilizing three rounds of amplification and hybridized to Affymetrix GeneChipâ Bovine Genome Expression Arrays (Affymetrix, Inc., Santa Clara, CA) utilizing Ambion’s MessageAmp TM II aRNA Amplification and MessageAmpTM II-Biotin Enhanced Kits (Applied Biosystems, Foster City, CA) according to the manufacturers’ instructions.
Hybridization protocol
Following a 16-hour hybridization the chip was washed and stained utilizing an Affymetrix GeneChipâ Fluidics Station 450 and Affymetrix GeneChipâ Command Console Software. Initial staining utilized streptavidin-conjugated phycoerythrin dye (Invitrogen Corporation, Carlsbad, CA). This stain was enhanced with biotinylated goat anti-streptavidin antibody (Vector Laboratories, Burlingame, CA), then counterstained with the conjugated phycoerythrin.
Scan protocol
The chip was scanned with an Affymetrix GeneChipâ Scanner model 3000 7G Plus. Files of the fluorescence intensities were utilized to generate corresponding probe expression levels utilizing Affymetrix Expression Console version 1.1, and quality control values were checked.
Description
Gene expression data from morula
Data processing
The data were analyzed with dChip. Normalization was under default settings.
