|
| Status |
Public on Oct 27, 2008 |
| Title |
Functionally-regressed Late luteal phase CL, biological rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Corpus luteum collected between days 14-16 after the midcycle LH surge from animal with serum concentrations of progesterone <0.5 ng/ml at time of CL collection
|
| Organism |
Macaca mulatta |
| Characteristics |
Gender:female, Tissue: Corpus luteum, reproductive state:normal menstrual cyclicity
|
| 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. Following the midcycle estradiol surge, the first day of low serum estradiol (< 100pg/ml) corresponds with the day after the LH surge (LH surge = day 0). On the appropriate day following the midcycle LH surge, the CL was collected during aseptic midline laparotomy, immediately flash frozen in liquid nitrogen, and stored at -80°C until total RNA was extracted.
|
| 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 that are undergoing functional regression. Luteolysis (CL death) is subdivided into two steps: 1) a decrease in progesterone secretion (functional regression), and 2) apoptosis and structural remodeling of luteal tissue (structural regression). This stage corresponds to CL in the first stage of luteolysis (functional regression). They have, or are about to, quit producing progesterone, but the tissue has not yet started to degenerate.
|
| 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 |
Sep 16, 2008 |
| Last update date |
Oct 27, 2008 |
| Contact name |
Randy Lloyd Bogan |
| E-mail(s) |
boganr@ohsu.edu
|
| Phone |
(503)614-3751
|
| Fax |
(503)690-5563
|
| Organization name |
Oregon National Primate Research Center
|
| Department |
Reproductive Sciences
|
| Lab |
Hennebold Lab
|
| Street address |
505 NW 185th Ave
|
| City |
Beaverton |
| State/province |
OR |
| ZIP/Postal code |
97123 |
| Country |
USA |
| |
|
| Platform ID |
GPL3535 |
| Series (1) |
| GSE12807 |
Gene expression data throughout spontaneous functional regression of the rhesus macaque corpus luteum. |
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