|
| Status |
Public on Feb 15, 2011 |
| Title |
12-2 M.mulatta Ovul Follicle 12h post-hCG |
| Sample type |
RNA |
| |
|
| Source name |
Follicle collected at 12 hour post-hCG treatment
|
| Organism |
Macaca mulatta |
| Characteristics |
gender: female
tissue: Follicle
reproductive state: controlled ovulation
|
| Biomaterial provider |
Oregon National Primate Research Center
|
| Treatment protocol |
Blood samples were collected daily by saphenous venipuncture starting six days after onset of menses. Serum was separated and samples were assayed daily for estradiol concentrations. When estradiol levels reached 100 ~ 120 pg/ml, a GnRH antagonist (Acyline, 0.1 ml/kg) plus recombinant (r)-human (h) FSH and r-hLH (30 IU each) were administered at 16:00 that day (day 1) and at 08:00 next day (day 2). At 16:00 on day 2, 1000 IU r-hCG was injected to initiate ovulatory events. The ovary bearing the large dominant follicle was removed from anesthetized animals by laparotomy at 12 h post-hCG treatment, and the follicle was separated from the ovary, immediately frozen in liquid nitrogen, and stored at -80¡ãC until total RNA was extracted. Daily blood samples were collected until the day of surgery.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol (Invitrogen) extraction of total RNA was performed according to the manufacturer's instructions, and total RNA obtained by Trizol extraction was further purified using RNeasy spin columns (Qiagen) following manufacturer instructions.
|
| Label |
Biotin
|
| Label protocol |
Messenger RNA is amplified and labeled from 2 micrograms of total RNA in two steps according to the standard Affymetrix one-cycle cDNA protocol (Expression Analysis Technical Manual Rev.4). In the first step, mRNA is converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix). In the second step, amplified and labeled cRNA (the target) is produced in an in vitro transcription reaction using T7 RNA polymerase and biotin-UTP (Affymetrix). Following removal of free nucleotides, target yield is measured by UV260 absorbance.
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| |
|
| Hybridization protocol |
Labeled target is fragmented at 95C in the presence of high magnesium concentration. The fragmented material is combined with biotinylated hybridization control oligomer and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Ten micrograms of target is hybridized for 16 hours at 45C to the GeneChip Rhesus Macaque Genome array (Affymetrix), followed by washing, staining with streptavidin-phycoerythrin (Molecular Probes), signal amplification with biotinylated anti-streptavidin antibody (Vector Labs), and a final staining step on the Fluidics Station 450 (Affymetrix).
|
| Scan protocol |
GeneChips were scanned using the GeneChip Scanner 3000 with the 7G upgrade (Affymetrix) and GCOS version 1.4.0 software (Affymetrix), yielding cell fluorescence intensity (.cel files). Image inspection was performed manually immediately following each scan.
|
| Description |
Gene expression data from CL during luteal development (luteinization), i.e. just after ovulation when the cellular constituents remaining from the preovulatory follicle are transformed into the CL.
|
| Data processing |
The processed image files (.cel) were normalized across arrays using the robust multichip average (RMA) algorithm and log-transformed (base 2).After normalization, GeneSifter (VizX Labs) microarray expression analysis software was used to analyze the resultant data.
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| |
|
| Submission date |
Jul 06, 2010 |
| Last update date |
Feb 15, 2011 |
| Contact name |
fuhua xu |
| E-mail(s) |
[email protected]
|
| Organization name |
Oregon National Primate Research Center
|
| Street address |
505 NW 185th Ave
|
| City |
Beaverton |
| State/province |
OR |
| ZIP/Postal code |
97006 |
| Country |
USA |
| |
|
| Platform ID |
GPL3535 |
| Series (1) |
| GSE22776 |
Dynamics of the Transcriptome in the Primate Ovulatory Follicle |
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