|
| Status |
Public on Dec 01, 2008 |
| Title |
60 in vivo developed blastocysts at H90 pc from 7 female rabbits 8BIV3.a2/4F1 |
| Sample type |
RNA |
| |
|
| Source name |
60 in vivo developped Blastocysts at H90 pc coming from 7 female rabbits
|
| Organism |
Oryctolagus cuniculus |
| Characteristics |
Embryo stage: Expanded blastocyst
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted from batches of embryos using the Rneasy Mini Kit (Qiagen, CA, USA). Purification procedure include a DNase I treatment (37°C, 30 min). In vitro transcription was carried out from an aliquot of the total extract equivalent to the desired number of embryos, using the MessageAmp aRNA Kit (Ambion) according to manufacturer's instructions.
|
| Label |
P33
|
| Label protocol |
One µg aRNA was mixed with 1µg of random hexamers (Promega) in a volume of 50 µl then incubated at 70°C for 10 min. cDNA was synthesised by the addition of 20 µl first-strand buffer 5X, 10µl 0.1M DTT, 5µl 10 mM dNTPs (-dATP), 10µl [~-33P] dATP (Perkin Elmer), 400U Superscript II (Invitrogen) and incubation at 25°C 10 min, 42°C 50 min, 70°C 15 min. The RNA template was removed by the addition of 2 µl RNAse H and incubation at 37°C for 20min. aRNA labelled probes were then purified on G-50 Sephadex columns.
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| |
|
| Hybridization protocol |
Prehybridization of the nylon micro-arrays were performed in 12 ml glass tubes with 1 ml ExpressHyb Hybridization Solution (Clontech) at 68 °C during 44 hours. The all purified labelled target (50 µl) were added to the prehybridization solution and allowed to hybridyse for 24 hours at 68 °C. Washing were performed 4 times in 2X SSC, 1% SDS and once in 0.1X SSC, 0.5% SDS at 68 °C for 30 min each.
|
| Scan protocol |
Scanner:BAS-5000 Fuji - Feature extraction software: AGScan
|
| Description |
Hybridization of labeled antisens RNA products
|
| Data processing |
The initial values of the hybridised signals were obtained after image acquisition using the AGScan software. Signals from negative and positive controls on the array were then zero. Decimal log values were calculated, then normalized by the mean signal value on the array without taking into account the zero values. Transformed values from negative and positive controls on the array were then zero and then all values were normalized by standard deviation.
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| |
|
| Submission date |
Jul 11, 2008 |
| Last update date |
Jul 17, 2008 |
| Contact name |
Duranthon Veronique |
| E-mail(s) |
[email protected]
|
| Phone |
33 1 34 65 25 92
|
| Organization name |
INRA
|
| Department |
Biologie du Développement et Reproduction
|
| Lab |
Equipe Plasticité du Génome
|
| Street address |
Domaine de Vilvert
|
| City |
JOUY EN JOSAS |
| ZIP/Postal code |
78350 |
| Country |
France |
| |
|
| Platform ID |
GPL7019 |
| Series (1) |
| GSE12084 |
In vivo rabbit preimplantation development |
|
