|
| Status |
Public on Dec 01, 2008 |
| Title |
60 in vivo developed blastocysts at H90 pc from 7 female rabbits 8BIV3.p2/4F2 |
| 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) according to manufacturer's instructions. Purification procedure include a DNase I treatment (37°C, 30 min). A PCR amplification method was carried out as described by Pacheco-Trigon et al. (2002) from an aliquot of the total extract equivalent to the desired number of embryos. Briefly, total RNAs were incubated with 0,087microl diluted Tc-primer (7,35microg/ml), 0,2microl 1 mM dNTPs, 0,25microl 10% NP40, 0,25microl 0.1M DTT, 0,93microl first-strand buffer 5X, 10U RNAse inhibitor (Roche) at 65°C for 2 min, at room temperature for 3 min then cooled on ice. cDNAs were synthesised by the addition of 100U Superscript II (Invitrogen) and 1.2U AMV (Invitrogen) and incubation at 42°C for 30 min. First-strand cDNA wer poly(dG)-tailed by incubation with 0.4microl 20mM dGTP, 2microl TdT buffer 5X, 1.6 microl water and 1microl TDT enzyme (Promega) at 37°C for 1 hour. The totality of reverse transcription - tailing product(10microl) was subjected for further two rounds of PCR amplification. PCR were performed in a total volume of 50 microl using 15 units of Goldstar DNA polymerase (Eurogentec) for each round of PCR cycles. Samples were incubated at 94°C for 10 min before two rounds of PCR (18 and 18 for the first and second PCR): 94°C for 2 min; 63°C for 50 sec and 72°C for 6 min. PCR products were purified using the QIAquick PCR Purification Kit and then used for labelling.
|
| Label |
P33
|
| Label protocol |
Five hundred cDNA from PCR products were labeled with [alpha-33P] dATP (Perkin Elmer) using the Atlas SMART Probe Amplification Kit (Clontech) according to manufacturuer's instruction.cDNA 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 microl) 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 PCR 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 |
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