ovaries dissected from 4-day old Sprague-Dawley rats and cleaned of ovarian bursa and cultured for 10 days with AMH
Treatment protocol
Female day P4 rat pups were euthanized and rat ovaries were removed according to IACUC-approved animal use protocols. Ovaries were placed in culture for ten days with treatment of 50ng/mL AMH. Whole ovaries were cultured as previously described (Nilsson, Parrott et al. 2001) on floating filters (0.4 um Millicell-CM, Millipore, Bedford, MD) in 0.5 ml Dulbecco's Modified Eagle's Medium (DMEM)-Ham's F-12 medium (1:1, vol/vol) containing 0.1 % bovine serum albumin (BSA, Sigma, St. Louis, MO), 0.1% Albumax (Gibco BRL, Gaithersburg, MD), 27.5 ug/ml transferrin, 200 ug/ml insulin (human recombinant, Sigma), and 0.05 ug/ml L-ascorbic acid (Sigma) in a 4-well culture plate (Nunc plate, Applied Scientific, South San Francisco, CA). Medium was supplemented with penicillin and streptomycin to prevent bacterial contamination.
Extracted molecule
total RNA
Extraction protocol
About 10 ovaries with the same treatment were pooled to make each RNA sample. RNA was extracted using the Trizol reagent according to manufacturer's instructions (Sigma, St. Louis, MO). Two independent RNA samples were analysed for each treatment group.
Label
biotin
Label protocol
standard Affymetrix procedures
Hybridization protocol
standard Affymetrix procedure
Scan protocol
chips were scanned using Agilent GeneArray Scanner G2500A
Description
Chip ID=Eric-MIS-2-092805. RNA from P4-day old rat ovaries cultured with AMH for 10 days, replicate 2
Data processing
Auto-scale ON (1-1000). Scaling = all probe sets. The microarray image data were converted to numerical data with GeneChip Operating Software using a scaling factor of 125, and then imported into the GeneSpring program (Agilent Technologies, Palo Alto, CA).
