|
| Status |
Public on Mar 31, 2016 |
| Title |
Cy5 54 Yg Vs Cy3 9 Og |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Growing_Follicle_Young
|
| Organism |
Bos taurus |
| Characteristics |
reproductive status (cattle): Heifer/nulliparous
|
| Growth protocol |
Aged (n=3) and young (n=3) Hereford beef cows were synchronized for ovualtion by giving a single intramuscular injection of PGF2α (Lutalyse® @ 25mg, Pfizer). Ovualtion and subsequent follicular dynamics were examined daily by transrectal ultrasonography using a 7.5 MHz linear-array transducer (Aloka SD 900, Tokyo, Japan). The day of ovulation was taken as the day of emergence of the first follicular wave (Day 0) [34, 35]. Granulosa cells were harvested on Day 3 (i.e., by ovariectomy or transvaginal aspiration). Follicular fluid was aliquoted into two RNase free 1.5 ml tubes after searching and removing the oocyte. Granulosa cells were obtained by centrifuging follicular fluid at 700g for 5 minutes. Follicular fluid was removed from both aliquots and granulosa cells were either snap frozen in liquid nitrogen or suspended in 300-700 µl of RNAlater® (Ambion Inc.) for 12 hours at 4°C. All samples were kept at -80°C until the extraction of RNA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
PicoPure RNA Isolation Kit followed by RiboAmpb HSPlus RNA Amplification kit (Molecular devices).
|
| Label |
Cy5
|
| Label protocol |
aRNA was labeled 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 |
Growing_Follicle_Old
|
| Organism |
Bos taurus |
| Characteristics |
reproductive status (cattle): Multiparity
|
| Growth protocol |
Aged (n=3) and young (n=3) Hereford beef cows were synchronized for ovualtion by giving a single intramuscular injection of PGF2α (Lutalyse® @ 25mg, Pfizer). Ovualtion and subsequent follicular dynamics were examined daily by transrectal ultrasonography using a 7.5 MHz linear-array transducer (Aloka SD 900, Tokyo, Japan). The day of ovulation was taken as the day of emergence of the first follicular wave (Day 0) [34, 35]. Granulosa cells were harvested on Day 3 (i.e., by ovariectomy or transvaginal aspiration). Follicular fluid was aliquoted into two RNase free 1.5 ml tubes after searching and removing the oocyte. Granulosa cells were obtained by centrifuging follicular fluid at 700g for 5 minutes. Follicular fluid was removed from both aliquots and granulosa cells were either snap frozen in liquid nitrogen or suspended in 300-700 µl of RNAlater® (Ambion Inc.) for 12 hours at 4°C. All samples were kept at -80°C until the extraction of RNA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
PicoPure RNA Isolation Kit followed by RiboAmpb HSPlus RNA Amplification kit (Molecular devices).
|
| Label |
Cy3
|
| Label protocol |
aRNA was labeled 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 |
Hybridization of microarray slides 4x44K printed with oligonucleotides by Agilent for all EmbryoGENE related projects.
|
| Scan protocol |
Images were scanned using Tecan scanning protocol.
|
| Data processing |
Arraypro software was used to extract target intensities. Background of a target was subtracted from the median signal intensity of the foreground of the target. If the difference was a negative value due to higher intensity of background vs. foreground signals, the negative value was replaced with a default value of 0.5. Flexarry software (1.6.1.1) was used to obtain the list of differentially expressed gene. The median pixel value for each target was transformed to the log2 and normalized “within array” for dye bias using nonparametric regression (locally weighted scatter plot smoothing; “lowess”), and subjected to “between-array” normalization to unify intensities across the arrays. Finally, a list of differentially expressed genes was obtained by running a simple linear model for microarray data; i.e., “simple limma algorithm”. A gene was considered to be differentially expressed when a ≥ 2-fold change in its expression at P-value of ≤ 0.05.
|
| |
|
| Submission date |
Nov 18, 2013 |
| Last update date |
Mar 31, 2016 |
| Contact name |
Muhammad Irfan-ur-Rehman Khan |
| E-mail(s) |
irfan.khan@usask.ca
|
| Organization name |
University of Saskatchewan
|
| Department |
Veterinary Biomedical Sciences
|
| Lab |
Jaswant Singh
|
| Street address |
52 Campus drive
|
| City |
SASKATOON |
| State/province |
SASKATCHEWAN |
| ZIP/Postal code |
S7N5B4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13226 |
| Series (1) |
| GSE52480 |
Maternal age related changes in transcriptome of bovine granulosa cells of the dominant follicle at the time of selection |
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