|
| Status |
Public on Mar 11, 2011 |
| Title |
Blastocysts in vivo biologial 2 rep2 |
| Sample type |
RNA |
| |
|
| Source name |
In vivo collected blastocysts by uterine flushing on day 7.0 post insemination
|
| Organism |
Bos taurus |
| Characteristics |
source: In vivo
time of collection: Day 7.0 post insemination
|
| Treatment protocol |
Treatments consists of the different in vitro production systems used. Blastocysts were produced in a total of ten different in vitro systems. In vivo blastocysts were also included and used as reference.
|
| Growth protocol |
Bovine ovaries were collected from the slaughterhouse, cumulus-oocyte complexes were aspirated from 3-6 mm follicles. Selection was done on general morphology and only COCs containing at least 5 layers of cumulus cells were kept. The selected COCs were washed in TLH medium and put through in vitro maturation, in vitro fertilization and in vitro culture. Blastocysts were collected on day 7.5 of culture. For in vivo counterparts, the embryos were collected 7.0 days post insemination. The embryos were washed in PBS, pooled in groups of 10 with mininal volume of liquid and kept à -80C until total RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using PicoPure columns following the manufacturer's recommandations. A DNAse 1 treatment was performed on column to remove contaminating genomic DNA.
|
| Label |
Alexa 647
|
| Label protocol |
RNA samples were amplified using the RiboAmp kit (Molecular Devices). Protocol was performed as recommanded to the exception that during the second round of amplification, amino-allyl coupled nucleotides were added to the IVT mix. An aliquot of 5 µg of aRNA was labelled by chemical addition of the Alexa 647 dye. Unincorporated dyes were cleaned up using Clean Up RNeasy Minikit columns (Qiagen).
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| |
|
| Hybridization protocol |
Labelled samples were concentrated by isopropanol precipitation and pellets were re-suspended in 5 µl of RNAse-free water. The concentrated labeled samples were mixed with 80 µl of hybridization buffer (HybBuffer #1 from Ambion) and deposited on the microarray under a LifterSlip coverslip (Thermo Scientific, Mississauga, ON, Canada). Microarray incubation was conducted for 21 h at 50˚C in an automated station (the Slidebooster apparatus from Advalytix, Danvers, MA). The coverslips were then gently removed in a low stringency buffer (2X SSC + 0.5X SDS). Slides were washed twice in the low stringency buffer for 15 min at 50˚C, transferred to high stringency buffer (0.5X SSC + 0.5X SDS) and washed twice for 15 min at 50˚C and finally dipped quickly in a 1.25X SSC solution and dried by centrifugation at 1,200xg for 5 min at room temperature.
|
| Scan protocol |
Slides were read on a VersArray ChipReader (Bio-Rad, Mississauga, ON, Canada). Image analysis was performed using ArrayPro software version 4.5 (MediaCybernetics, Bethesda, MD). For each spot, background was determined locally. Data tables containing the foreground, background and flagged spot annotation were generated for downstream data processing and
|
| Description |
Treatment #1 of publication
|
| Data processing |
Microarray data were pre-processed as follows: 1) background correction was conducted by simple subtraction; 2) mean values were calculated for the technically duplicated arrays; 3) median values were calculated for technically duplicated features spotted on the microarrays; 4) within-array normalization was performed with Loess; 5) Quantile was applied for inter-array normalization; 6) the entire dataset was trimmed according to a cut-off value calculated from the mean values of selected negative control features (Aliens, GFP and Arabidopsis) plus two standard deviations. Steps 3 to 5 as well as the statistical analysis and all downstream steps were conducted in WebArrayDB (Xia et al. 2005, Wang et al. 2009): http://www.webarraydb.org/ webarray/index.html.
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| |
|
| Submission date |
Mar 09, 2011 |
| Last update date |
Mar 11, 2011 |
| Contact name |
Claude Robert |
| E-mail(s) |
claude.robert@fsaa.ulaval.ca
|
| Organization name |
Laval University
|
| Department |
Animal Sciences
|
| Street address |
Pavillon Comtois
|
| City |
Quebec |
| State/province |
Quebec |
| ZIP/Postal code |
G1V0A6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13261 |
| Series (1) |
| GSE27872 |
Comprehensive across production systems assessment of the impact of in vitro micro-environment on the expression of messengers and long non-coding RNAs in the bovine blastocyst |
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