|
| Status |
Public on Sep 23, 2011 |
| Title |
Control – Vitrified 6 day old Embryos Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Vitrified 6 day old Embryos Replicate 2
|
| Organism |
Oryctolagus cuniculus |
| Characteristics |
strain: New Zealand White Rabbit
tissue: blastocyst
age: 6 days
treatment: Vitrified
|
| Treatment protocol |
Vitrification: The vitrifcation procedure was carried out in two steps at 20oC. In the frst step, embryos were placed for 2 min in a vitrifcation solution consisting of 12.5% (v/v) dimethyl sulphoxide (3.5 M DMSO, Sigma) and 12.5% (v/v) ethylene glycol (4.4 M EG, Sigma) in DPBS supplemented with 0.2% (w/v) of BSA. In the second step, embryos were suspended for 1 min in a solution of 20% (v/v) DMSO and 20% (v/v) EG in DPBS supplemented with 0.2% (w/v) of BSA. Then embryos suspended in vitrifcation medium were loaded into 0.25 ml plastic straws and two sections of DPBS were added at either end of each straw separated by air bubbles. Finally, straws were sealed and plunged directly into liquid nitrogen. Devitrifcation was performed by immersing the central and final sections of the straws in a water bath at 20oC for 10-15 sec. The vitrifcation medium was eliminated employing a solution of DPBS with 0.33 M sucrose for 5 min and a wash in a solution of DBPS for another 5 min.
Slow-freezing: Embryos were placed successively for 5 min into three different solutions consisting, respectively, of 0.5M, 1M and 1.5M dimethyl sulfoxide (DMSO) in DPBS supplemented with BSA. Then, the embryos suspended in the cryopreservation medium were oaded into 0.125 mL sterile plastic straws, and submited to slow freezing 1oC/min up to -7oC. Five minutes later manual seeding were done, and then embryos were cooled at 0.3oC/min up to -35oC. After that, the straws were plunged directly into liquid nitrogen. Thawing was performed by placing the straws 10 sec in air at room temperature and then in a water bath at 20oC for 1 min. The cryopreservation medium was removed in three steps of 5 min, in which embryos were placed successively in three wash solutions consisting of 1 M, 0.5 M and 0M DMSO in DPBS supplemented with BSA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Total RNA (100 ng) was labelled using QuickAmp Labelling Kit (Agilent Technologies) following manufacturer's instructions. Excess dye was removed with the RNAeasy Mini Kit (QIAGEN) and dye incorporation and concentration were determined using the microarray setting on the Nanodrop 1000.
|
| |
|
| Channel 2 |
| Source name |
Control 6 day old Embryos Replicate 2
|
| Organism |
Oryctolagus cuniculus |
| Characteristics |
strain: New Zealand White Rabbit
tissue: blastocyst
age: 6 days
treatment: Untreated
|
| Treatment protocol |
Vitrification: The vitrifcation procedure was carried out in two steps at 20oC. In the frst step, embryos were placed for 2 min in a vitrifcation solution consisting of 12.5% (v/v) dimethyl sulphoxide (3.5 M DMSO, Sigma) and 12.5% (v/v) ethylene glycol (4.4 M EG, Sigma) in DPBS supplemented with 0.2% (w/v) of BSA. In the second step, embryos were suspended for 1 min in a solution of 20% (v/v) DMSO and 20% (v/v) EG in DPBS supplemented with 0.2% (w/v) of BSA. Then embryos suspended in vitrifcation medium were loaded into 0.25 ml plastic straws and two sections of DPBS were added at either end of each straw separated by air bubbles. Finally, straws were sealed and plunged directly into liquid nitrogen. Devitrifcation was performed by immersing the central and final sections of the straws in a water bath at 20oC for 10-15 sec. The vitrifcation medium was eliminated employing a solution of DPBS with 0.33 M sucrose for 5 min and a wash in a solution of DBPS for another 5 min.
Slow-freezing: Embryos were placed successively for 5 min into three different solutions consisting, respectively, of 0.5M, 1M and 1.5M dimethyl sulfoxide (DMSO) in DPBS supplemented with BSA. Then, the embryos suspended in the cryopreservation medium were oaded into 0.125 mL sterile plastic straws, and submited to slow freezing 1oC/min up to -7oC. Five minutes later manual seeding were done, and then embryos were cooled at 0.3oC/min up to -35oC. After that, the straws were plunged directly into liquid nitrogen. Thawing was performed by placing the straws 10 sec in air at room temperature and then in a water bath at 20oC for 1 min. The cryopreservation medium was removed in three steps of 5 min, in which embryos were placed successively in three wash solutions consisting of 1 M, 0.5 M and 0M DMSO in DPBS supplemented with BSA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Total RNA (100 ng) was labelled using QuickAmp Labelling Kit (Agilent Technologies) following manufacturer's instructions. Excess dye was removed with the RNAeasy Mini Kit (QIAGEN) and dye incorporation and concentration were determined using the microarray setting on the Nanodrop 1000.
|
| |
|
| |
| Hybridization protocol |
Hibridization was done using Agilent Gene Expression Hybridization Kit, following manufacturer's instructions. After 17 hours at 65ºC on a , hybridised slides were washed sequential.
|
| Scan protocol |
Scanned on an Agilent G2505B scanner.
Images were quantified using Agilent Feature Extraction Software (version 10.10.1.1).
|
| Description |
Biological Replicate 2 of 3, dye swap, Control embryos vs. Vitrified embryos
|
| Data processing |
Agilent Feature Extraction Software (v 10.10.1.1) was used for background correction and LOWESS normalization.
|
| |
|
| Submission date |
Sep 20, 2011 |
| Last update date |
Sep 23, 2011 |
| Contact name |
M. Desemparats Saenz-de-Juano |
| E-mail(s) |
masaede@upvnet.upv.es
|
| Organization name |
Universidad Politécnica Valencia
|
| Department |
Animal Science
|
| Lab |
Biotechnology of Reproduction
|
| Street address |
Camino de Vera s/n
|
| City |
Valencia |
| State/province |
Valencia |
| ZIP/Postal code |
46022 |
| Country |
Spain |
| |
|
| Platform ID |
GPL7083 |
| Series (1) |
| GSE32263 |
Effect of cryopreservation on rabbit late blastocyst transcriptome |
|
