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Sample GSM456640 Query DataSets for GSM456640
Status Public on Feb 20, 2010
Title bovine embryo at morula stage, biological rep 2
Sample type RNA
 
Source name Bovine morula
Organism Bos taurus
Characteristics development stage: morula
Treatment protocol Human embryos: Supernumerary human embryos were obtained from patients at the Boston IVF Clinic through informed consent with the approval of the Internal Review Board of Harvard University. Embryos thus obtained were cultured in a two-step culture system (Sage/Biopharma, CT) in micro-drops at 37? C and 5% CO2 under oil (Sage, CT) as described previously. Embryos from each stage were segregated into three groups. Each group was considered to be a biologic replicate: embryos within each replicate were pooled for further analysis. The blastocyst embryos were graded according to the Gardner scale. Mouse embryos: Briefly, female mice were impregnated, and embryos were flushed from the oviducts. Individual embryos were morphologically staged by light microscopy. Bovine embryos: Ovaries were washed and 2-6 mm follicles were aspirated. 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 we placed maturation media that had been equilibrated in 5% carbon dioxide and air (39°C, high humidity). The 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 μl 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, 2.0μg heparin (Sigma H-3125), pencillamine, hypotaurine and epinephrine (Sigma-P-4875, H-1384, E-4250) to each drop of fertilization media. Oocytes were removed from the 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 50μl drop of SOF culture medium, supplemented with 8 mg/ml of fatty acid-free BSA, nonessential and essential amino acids and pyruvate. The IVF conditions were optimized for each species. To minimize the influence of IVF conditions to cross-species comparison, we subsequently based the comparisons on either relative expression changes or co-expression patterns.
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.
 
Submission date Sep 25, 2009
Last update date Feb 20, 2010
Contact name Dan Xie
E-mail(s) danxie2@uiuc.edu
Organization name University of Illinois at Urbana and Champaign
Department Bioengineering
Street address 3215 Digital Computer Lab, MC-278, 1304 W. Springfield Ave
City Urbana
State/province IL
ZIP/Postal code 61801
Country USA
 
Platform ID GPL2112
Series (1)
GSE18290 Time course expression data from early Bovine embryo, Human embryo, and Mouse embryo

Data table header descriptions
ID_REF
VALUE dChip signal intensity

Data table
ID_REF VALUE
AFFX-BioB-5_at 328.9
AFFX-BioB-M_at 318.57
AFFX-BioB-3_at 249.8
AFFX-BioC-5_at 848.02
AFFX-BioC-3_at 844.56
AFFX-BioDn-5_at 2657.28
AFFX-BioDn-3_at 2996.94
AFFX-CreX-5_at 12430.49
AFFX-CreX-3_at 12746.75
AFFX-DapX-5_at 97.76
AFFX-DapX-M_at 135.9
AFFX-DapX-3_at 232.53
AFFX-LysX-5_at 23.4
AFFX-LysX-M_at 29.83
AFFX-LysX-3_at 59.86
AFFX-PheX-5_at 45.54
AFFX-PheX-M_at 34.27
AFFX-PheX-3_at 50.82
AFFX-ThrX-5_at 18.07
AFFX-ThrX-M_at 140.05

Total number of rows: 24128

Table truncated, full table size 543 Kbytes.




Supplementary file Size Download File type/resource
GSM456640.CEL.gz 2.1 Mb (ftp)(http) CEL
Processed data included within Sample table

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