|
| Status |
Public on Oct 26, 2018 |
| Title |
Array 3_Cum_68h-Cetro |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Cumulus cells_Cetrorelix
|
| Organism |
Bos taurus |
| Characteristics |
breed (cattle): holstein
cell type: Cumulus cells
time: 68 hours
treatment: Cetrorelix 12mg/day
|
| Treatment protocol |
six dairy cows were stimulated with FSH given twice daily for three days followed by an FSH withdrawal period of 68h « 68h condition ». For the « 68h +Cetro » condition, the GnRH agonist Cetrorelix (Cetrotide, Merckx-Serono), which inhibits LH secretion, was given from day 2 of FSH stimulation until COC recovery 68h following the last FSH injection (12mg per day for 5 days).
collect protocol: The recovered COC were washed twice in TLH solution and were then collected in Petri dishes containing PBS and polyvinyl alcohol (PVA). The COC were washed three times with PBS-PVA in different dishes, transferred into 1.5 ml conical tubes, and vortexed in 150µl PBS for 2min. Cells were placed into a new dish to collect oocytes and cumulus cells (CC) separately into new 1.5-ml conical tubes. The CC were washed by centrifugation three times in 150µl PBS. Cells were then snap frozen in liquid nitrogen and conserved at 80°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction using the PicoPure RNA Isolation Kit from Thermo Fisher.
|
| Label |
Cy5
|
| Label protocol |
amplification of RNA using the RiboAmpb HSPlus RNA Amplification kit (Thermo Fisher).
The labelling aRNA was done using the Universal Linkage System (ULS) by Kreatech for Gene Expression studies using microarray slides from Agilent. ULS is a non-enzymatic protocol that allows direct labeling of unmodified, amplified RNA. ULS will react with unmodified, amplified RNA and label it by formation of a coordinative bond on the N7 position of guanine. Fluorophore Cy3 and Cy5 are linked via a spacer to the ULS molecule.
|
| |
|
| Channel 2 |
| Source name |
Cumulus cells
|
| Organism |
Bos taurus |
| Characteristics |
breed (cattle): holstein
cell type: Cumulus cells
time: 68 hours
treatment: none
|
| Treatment protocol |
six dairy cows were stimulated with FSH given twice daily for three days followed by an FSH withdrawal period of 68h « 68h condition ». For the « 68h +Cetro » condition, the GnRH agonist Cetrorelix (Cetrotide, Merckx-Serono), which inhibits LH secretion, was given from day 2 of FSH stimulation until COC recovery 68h following the last FSH injection (12mg per day for 5 days).
collect protocol: The recovered COC were washed twice in TLH solution and were then collected in Petri dishes containing PBS and polyvinyl alcohol (PVA). The COC were washed three times with PBS-PVA in different dishes, transferred into 1.5 ml conical tubes, and vortexed in 150µl PBS for 2min. Cells were placed into a new dish to collect oocytes and cumulus cells (CC) separately into new 1.5-ml conical tubes. The CC were washed by centrifugation three times in 150µl PBS. Cells were then snap frozen in liquid nitrogen and conserved at 80°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction using the PicoPure RNA Isolation Kit from Thermo Fisher.
|
| Label |
Cy3
|
| Label protocol |
amplification of RNA using the RiboAmpb HSPlus RNA Amplification kit (Thermo Fisher).
The labelling aRNA was done using the Universal Linkage System (ULS) by Kreatech for Gene Expression studies using microarray slides from Agilent. ULS is a non-enzymatic protocol that allows direct labeling of unmodified, amplified RNA. ULS will react with unmodified, amplified RNA and label it by formation of a coordinative bond on the N7 position of guanine. Fluorophore Cy3 and Cy5 are linked via a spacer to the ULS molecule.
|
| |
|
| |
| Hybridization protocol |
The hybridization of microarray slides 4x44K printed with oligonucleotides by Agilent for all EmbryoGENE related projects using the expression array protocol.
|
| Scan protocol |
Scan on a PowerScanner (Tecan) using the autogain function. Images were quantified using ArrayPro version 6.3 (MediaCybernetics)
|
| Description |
Biological replicate 3 of 3
|
| Data processing |
Signal intensity data files were normalized and analyzed using the FlexArray 1.6.1 software (Genome Quebec, URL: http://genomequebec.mcgill.ca/FlexArray). The first step in the data processing was to remove the background of intensity files using a simple background subtraction. Data were then normalized for dye bias using a within-array loess and a between-array « quantile » normalization to minimize array effects. To calculate fold changes of probe intensity, normalized data were assessed using the e Bayes moderated t-test (LIMMA) included in the FlexArray software. A False Discovery Rate algorithm (Benjamini-Hochberg) was also applied.
|
| |
|
| Submission date |
Oct 25, 2018 |
| Last update date |
Oct 26, 2018 |
| 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 |
GPL13226 |
| Series (1) |
| GSE121783 |
The effects of basal LH inhibition with Cetrorelix on cumulus cell gene expression during the luteal phase under ovarian coasting stimulation in cattle. |
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