|
| Status |
Public on Jun 08, 2012 |
| Title |
BF178_IVP |
| Sample type |
RNA |
| |
|
| Source name |
reproduction mode: IVP
|
| Organism |
Bos taurus |
| Characteristics |
embryo name: BF178
Stage: embryonic
somatic tissue: no
reproduction mode: IVP
|
| Biomaterial provider |
INRA/UMR Biologie du Developpement et Reproduction
|
| Treatment protocol |
Tissues were collected and immediately snap-frozen. Total RNA from IVP and SCNT embryos were extracted with TrizolTM (Invitrogen). RNA quality was first verified by A260/280 absorbance ratios.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
IVT amplified RNA from each sample was synthesized with the MessageAmpTM aRNA Kit (Ambion) according to Degrelle et al., 2008 (RT on 1µg total RNA, IVT during 10h). aRNA was purified on Mini Quick Spin RNA columns (Roche Diagnostic).
|
| Label |
33P
|
| Label protocol |
aRNA was retro-transcribed and directly labelled with [alpha-33P]dATP as described (Degrelle et al., 2008). 500ng of aRNA was mixed with 500ng of random hexamers in a volume of 25µl. The mixture was incubated at 70°C for 10 min and chilled on ice. cDNA was synthesised by the addition of 5µl 10X PCR buffer, 5µl 25mM MgCl2, 5 µl 0,1 mM DTT, 2,5µl 10mM mix dGTP, dCTP and dTTP, 2,5µl water, 50 µCi [alpha-33P]dATP and 200U Superscript II (Invitrogen) at 42°C for 50min. The RNA template was removed by the addition of 1µl RNAse H- and incubation at 37°C for 20 min. Labelled targets were then purified on Sephadex columns (G-50).
|
| |
|
| Hybridization protocol |
Prehybridisation in ExpressHybTM Hybridization Solution (Clontech) at 68°C during 1h (14ml). Hybridisation using new ExpressHybTM Hybridization Solution (Clontech) at 68°C overnight (10ml). Arrays were washed four times in 2X SSC, 1% SDS and once in 0.1X SSC, 0.5% SDS at 68°C for 30 min each. They were then exposed to phosphor-screens (Amersham) for 5 days.
|
| Scan protocol |
Scanner: STORM 760 from Molecular Dynamics - Raw data set : Feature extraction with Imagene 5.1 software from BioDiscovery (Proteigene)
|
| Description |
Embryonic stage was defined by "Brachuyry" ISH on the embryonic disc (Hue et al. 2001). SCNT: somatic cell nuclear transfer. Donor cells: skin-ear fibroblasts from 3 different Holstein cows: 5538, 7711, 029. Syntax of the sample names: embryoName_ReproductionMode. Where the reproduction mode is either IVP, SCNT-0029, SCNT-771 and SCNT-5538.
|
| Data processing |
Intensity = Signal Mean - Normalization = Signal mean were log2 transformed and normalized by the mean of the all intensities on the array
|
| |
|
| Submission date |
Jan 09, 2012 |
| Last update date |
Jun 08, 2012 |
| Contact name |
Isabelle Hue |
| E-mail(s) |
isabelle.hue@jouy.inra.fr
|
| Phone |
33134652567
|
| Fax |
33134652364
|
| Organization name |
INRA
|
| Department |
PHASE
|
| Lab |
UMR Biologie du Developpement et Reproduction
|
| Street address |
Domaine de Vilvert
|
| City |
Jouy en Josas |
| ZIP/Postal code |
78350 |
| Country |
France |
| |
|
| Platform ID |
GPL7417 |
| Series (1) |
| GSE34944 |
Defective uncoupled differentiations after nuclear reprogramming |
|
