|
| Status |
Public on Feb 24, 2010 |
| Title |
Male Bull 2 sample 3 |
| Sample type |
RNA |
| |
|
| Source name |
Male blastocysts from Bull 2
|
| Organism |
Bos taurus |
| Characteristics |
cell type: blastocyst
genotype: Mixed breed
age: Day 7 post insemination
gender: Male
|
| Treatment protocol |
Blastocysts were snap frozen on Day 7 post-insemination and kept at -80 ºC until analysis.
|
| Growth protocol |
Details for embryo production are provided in Biology of Reproduction 2008; 79:594-597.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated and purified using RNaeasy Micro kit (Qiagen).
|
| Label |
biotin
|
| Label protocol |
Labelled cRNA was synthesized with a linear RNA amplification method (Message Amp Premier kit, Ambion) following provider´s instructions and using 50 ng of total RNA as template for reverse transcription.
|
| |
|
| Hybridization protocol |
Labelled cRNA was fragmented in fragmentation buffer (5× buffer: 200 mM Tris-acetate (pH 8.1)/500 mM KOAc/150 mM MgOAc) and hybridised to the microarrays in 200µl of hybridization solution containing 15µg labelled target in 1× Mes buffer (0.1 M Mes/1.0 M NaCl/20 mM EDTA 0.01%/Tween20) and 0.1mg/ml herring sperm DNA, 10% DMSO, 0.5mg/ml BSA, 50 pM control oligonucleotide B2 and 1x eukaryotic hybridization controls (bioB, bioC, bioD, cre). Both control oligonucleotide B2 and eukaryotic hybridisation controls were purchased from Affymetrix. Hybridization mix was applied to Bovine Genome array (Affymetrix), and arrays were placed on a rotisserie and rotated at 60 rpm for 16h at 45°C. Following hybridization, the arrays were washed and stained with a streptavidin-phycoerythrin conjugate (Molecular Probes).
|
| Scan protocol |
The arrays were scanned using a confocal scanner (GC3000_Affymetrix). The image data were analysed by GeneChip Operating Software (GCOS 1.4, Affymetrix)
|
| Description |
Gene expression data from male Day 7 blastocysts produced with sorted semen from Bull2
|
| Data processing |
For the Bioinformatic analysis dChip (www.dchip.org) and Affy/AffyPLM (Bioconductor) software were used to look for samples acting as outliers and Partek Genomics Suite 6.4 (Partek® software, Copyright © 2008 Partek Inc., St. Louis, MO, USA.Partek) to perform gene expression analysis. RMA processing was used for normalizing the data as well as a global median normalisation. The change in each gene expression was calculated by determining the fold change (ratio) of the mean intensity of each group.
|
| |
|
| Submission date |
Sep 01, 2009 |
| Last update date |
Feb 24, 2010 |
| Contact name |
Pablo Bermejo-Álvarez |
| E-mail(s) |
borrillobermejo@hotmail.com
|
| Phone |
0034-913473768
|
| Fax |
0034-913474014
|
| Organization name |
INIA
|
| Department |
Reproducción Animal
|
| Lab |
Molecular Embryology
|
| Street address |
Ctra La Coruña Km 5,9
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28039 |
| Country |
Spain |
| |
|
| Platform ID |
GPL2112 |
| Series (1) |
| GSE17921 |
Sex-related transcriptional differences in Day 7 bovine in vitro produced blastocysts |
|
