|
| Status |
Public on Apr 01, 2012 |
| Title |
Lame 58 Cy 3 Vivo Cy5 Ptv 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Embryo produced in vitro after OPU
|
| Organism |
Bos taurus |
| Characteristics |
sample type: in vitro embryos
time of embryo collection: 168 hpf (hours post-in-vitro fertilization)
in vitro production: Yes
|
| Treatment protocol |
For in-vitro: bovine ovaries were obtained from a slaughterhouse, cumulus-oocyte complexes aspiration were aspirated from 2 to 6 mm follicles. Healthy COCs with at least five layers of cumulus were selected for in vitro maturation, in vitro fertilization and in vitro culture. For In-vivo: On Day 8 to 12 post-oestrus, follicles with a diameter larger than 8 mm from Holstein heifers cows were aspirated. Thirty-six hours later, 8 injections of FSH were given for a total of 400 mg of FSH were administered in doses decreasing from 60 mg to 20 mg (two injections per day). A 500-µg dose of prostaglandin F2α analogue was administered with each of the two final FSH injections. The animals exhibited oestrus about 36 h after the final injection and were inseminated twice, 12 and 24 h post-estrus with the pooled semen used for in vitro fertilization. Embryos were recovered by uterine flushing 7.5 days after the first insemination.
|
| Growth protocol |
In vitro production
In vivo production
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extract using the PicoPure RNA Isolation Kit from Molecular Devices. The PicoPure RNA Isolation Kit enables to recover total cellular RNA from pico-scale samples. Researchers can obtain high recoveries of total cellular RNA from as little as a single cell to samples with up to 100 ug of RNA.
Amplification of RNA was done using the RiboAmpb HSPlus RNA Amplification kit (Molecular devices). Quantity of produce aRNA was determined using a Nanodrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA).
|
| Label |
Cy5
|
| Label protocol |
Samples were labeled using the ULS Fluorescent Labeling Kit for Agilent arrays (with Cy3 and Cy5) (Kreatech Diagnostics, Amsterdam, The Netherlands). The labeled product was then purified using the picopure RNA extraction kit (Applied Biosystems, Carlsbad, CA, USA) to remove uncoupled dyes.
|
| |
|
| Channel 2 |
| Source name |
In vivo
|
| Organism |
Bos taurus |
| Characteristics |
sample type: in vivo embryos
time of embryo collection: 7 dpi (days post-in-vivo insemination)
in vitro production: No
|
| Treatment protocol |
For in-vitro: bovine ovaries were obtained from a slaughterhouse, cumulus-oocyte complexes aspiration were aspirated from 2 to 6 mm follicles. Healthy COCs with at least five layers of cumulus were selected for in vitro maturation, in vitro fertilization and in vitro culture. For In-vivo: On Day 8 to 12 post-oestrus, follicles with a diameter larger than 8 mm from Holstein heifers cows were aspirated. Thirty-six hours later, 8 injections of FSH were given for a total of 400 mg of FSH were administered in doses decreasing from 60 mg to 20 mg (two injections per day). A 500-µg dose of prostaglandin F2α analogue was administered with each of the two final FSH injections. The animals exhibited oestrus about 36 h after the final injection and were inseminated twice, 12 and 24 h post-estrus with the pooled semen used for in vitro fertilization. Embryos were recovered by uterine flushing 7.5 days after the first insemination.
|
| Growth protocol |
In vitro production
In vivo production
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extract using the PicoPure RNA Isolation Kit from Molecular Devices. The PicoPure RNA Isolation Kit enables to recover total cellular RNA from pico-scale samples. Researchers can obtain high recoveries of total cellular RNA from as little as a single cell to samples with up to 100 ug of RNA.
Amplification of RNA was done using the RiboAmpb HSPlus RNA Amplification kit (Molecular devices). Quantity of produce aRNA was determined using a Nanodrop ND-1000 (NanoDrop Technologies, Wilmington, DE, USA).
|
| Label |
Cy3
|
| Label protocol |
Samples were labeled using the ULS Fluorescent Labeling Kit for Agilent arrays (with Cy3 and Cy5) (Kreatech Diagnostics, Amsterdam, The Netherlands). The labeled product was then purified using the picopure RNA extraction kit (Applied Biosystems, Carlsbad, CA, USA) to remove uncoupled dyes.
|
| |
|
| |
| Hybridization protocol |
825 ng of each labeled sample were incubated in a solution containing 2x blocking agent and 5x fragmentation buffer in a volume of 55 µl at 65°C for 15 minutes and were then put on ice. 55 µl of 2x GEx Hybridization Buffer HI-RPM was added for a final volume of 110 µl. The hybridization mix (100 µl) was added onto the array and hybridization was performed at 65°C for 17 h using an Agilent Hybridization chamber in a rotating oven. After the hybridization step, slides were washed with gene expression wash buffer 1 containing 0.005% triton X-102 for 3 minutes at room temperature and then transferred to gene expression wash buffer 2 containing 0.005% triton X-102 for 3 minutes at 42°C. Final washes with acetonitrile for 10 sec at room temperature and with drying and stabilisation solution for 30 sec at room temperature were performed before air-drying the slides.
|
| Scan protocol |
Slides were scan on Agilent G2505 B Micro Array Scanner.
|
| Data processing |
Images were quantified using Feature extraction software
|
| |
|
| Submission date |
Jan 11, 2012 |
| Last update date |
Apr 01, 2012 |
| Contact name |
Claude Robert |
| E-mail(s) |
claude.robert@fsaa.ulaval.ca
|
| Organization name |
Laval University
|
| Department |
Animal Sciences
|
| Street address |
Pavillon Comtois
|
| City |
Quebec |
| State/province |
Quebec |
| ZIP/Postal code |
G1V0A6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13226 |
| Series (1) |
| GSE35025 |
Transcriptome Analysis Of Bovine Expanded Blastocysts Produced In Vivo Vs In Vitro Sof Opu |
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