|
| Status |
Public on Dec 16, 2010 |
| Title |
Stage 3 BV48_st3 |
| Sample type |
RNA |
| |
|
| Source name |
Extra-embryonic tissues were immediately snap-frozen. Total RNA extracted with trizolTM (invitrogen). RNA quality verified by intact A260/280 absorbance ratios.
|
| Organism |
Bos taurus |
| Characteristics |
somatic tissue: no
embryo: yes
reproduction mode: AI
embryonic stage: 3
|
| Biomaterial provider |
INRA/UMR Biologie du Developpement et Reproduction
|
| Treatment protocol |
Tissues were collected and immediately snap-frozen. Total RNA from stage2 (n=5), stage3 (n=5), stage4 (n=5) and stage5+ (n=5) 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 1microg total RNA, IVT during 10h). aRNA was purified on Mini Quick Spin RNA columns (Roche Diagnostic).
|
| Label |
P33
|
| 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 25microl. The mixture was incubated at 70°C for 10 min and chilled on ice. cDNA was synthesised by the addition of 5microl 10X PCR buffer, 5microl 25mM MgCl2, 5 microl 0,1 mM DTT, 2,5microl 10mM mix dGTP, dCTP and dTTP, 2,5microl water, 50 microCi [alpha-33P]dATP and 200U Superscript II (Invitrogen) at 42°C for 50min. The RNA template was removed by the addition of 1microl 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 |
Syntax of the sample names: arraySlide_embryoName_embryonicStage Where the embryonic stage is either stage2, stage3, stage4 or stage5+
|
| Data processing |
Intensity = Signal Mean Normalization = Signal mean were log2 transformed and normalized by the mean of the all intensities on the array For the statistical analyses, described in the paper, the data was standardized by gene.
|
| |
|
| Submission date |
Oct 02, 2008 |
| Last update date |
Dec 16, 2010 |
| Contact name |
Isabelle Hue |
| E-mail(s) |
[email protected]
|
| 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) |
| GSE13013 |
Molecular prediction of gastrulation stages with small extra-embryonic gene-set |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
Normalized signal measurement for each hybridized spot |
| CH1_MEDIAN |
channel 1 signal intensity median - P33 |
| CH1_MEAN |
channel 1 signal intensity mean |
| CH1_SD |
channel 1 signal intensity standard deviation |
| CH1_TOTAL |
channel 1 signal intensity total |
| CH1_BKD_MEDIAN |
channel 1 background signal intensity median |
| CH1_BKD_MEAN |
channel 1 background signal intensity mean |
| CH1_BKD_SD |
channel 1 background signal intensity standard deviation |
| CH1_BKD_TOTAL |
channel 1 background signal intensity total |
