|
| Status |
Public on Feb 08, 2012 |
| Title |
Oocyte_GV versus Oocyte_MII |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Oocyte_GV
|
| Organism |
Bos taurus |
| Characteristics |
developmental stage: Germinal vesicule
|
| Growth protocol |
Follicles of 5 mm in diameter were aspirated from ovaries of slaughtered dairy cattle. The collected cumulus-oocyte complexes (COCs) were washed 4 times in TLH and selected to be placed in 50-µl drops of maturation medium covered with mineral oil. The oocytes at the germinal vesicle stage (GVs) were collected after the TLH washes. The COCs was matured in TCM-199 medium supplemented with 10% fetal calf serum (FCS), pyruvate and gentamycin. The dishes were placed for at 39°C under 5% CO2 with a maximum humidity level. After 22h of maturation, oocytes at MII stage were collected. All the GVs and MII collected were mechanically denuded by agitation for about 5 minutes in PBS to remove their cumulus cells before the freezing step.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using miRNeasy kit from Qiagen according to the manufacturer's protocol.
|
| Label |
Cy3
|
| Label protocol |
The microarray assay was performed using a service provider (LC Sciences). The assay started from 200 ng total RNA sample, which was amplified in-house then size fractionated using a YM-100 Microcon centrifugal filter (Millipore), and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent Cy3 dye staining
|
| |
|
| Channel 2 |
| Source name |
Oocyte_MII
|
| Organism |
Bos taurus |
| Characteristics |
developmental stage: metaphase II
|
| Growth protocol |
Follicles of 5 mm in diameter were aspirated from ovaries of slaughtered dairy cattle. The collected cumulus-oocyte complexes (COCs) were washed 4 times in TLH and selected to be placed in 50-µl drops of maturation medium covered with mineral oil. The oocytes at the germinal vesicle stage (GVs) were collected after the TLH washes. The COCs was matured in TCM-199 medium supplemented with 10% fetal calf serum (FCS), pyruvate and gentamycin. The dishes were placed for at 39°C under 5% CO2 with a maximum humidity level. After 22h of maturation, oocytes at MII stage were collected. All the GVs and MII collected were mechanically denuded by agitation for about 5 minutes in PBS to remove their cumulus cells before the freezing step.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using miRNeasy kit from Qiagen according to the manufacturer's protocol.
|
| Label |
Cy5
|
| Label protocol |
The microarray assay was performed using a service provider (LC Sciences). The assay started from 200 ng total RNA sample, which was amplified in-house then size fractionated using a YM-100 Microcon centrifugal filter (Millipore), and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent Cy3 dye staining
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies).
|
| Scan protocol |
Hybridization images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
|
| Description |
Each channel consists of one replicate with pools of oocytes.
|
| Data processing |
Raw intensities experienced the adjustments of data-filtering, log 2 transformation, gene centering and global normalization. MiRNAs with all samples intensities larger than the intensity threshold of 22 were considered detectable.
|
| |
|
| Submission date |
Feb 06, 2012 |
| Last update date |
Feb 08, 2012 |
| Contact name |
Marc-André Sirard |
| E-mail(s) |
Marc-Andre.Sirard@fsaa.ulaval.ca
|
| Organization name |
Université Laval
|
| Department |
Sciences Animales
|
| Street address |
Offfice 2732, 2440 Hochelaga Blvd.
|
| City |
Québec City |
| State/province |
Quebec |
| ZIP/Postal code |
G1V 0A6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL15194 |
| Series (1) |
| GSE35567 |
Analysis of microRNAs in bovine early embryonic development |
|
