strain: Sprague-Dawley gender: Female tissue: ovary developmental stage: day P4 incubated for 2 more days treatment group: 50 ng/ml AMH for 48 hours
Treatment protocol
For each ovary sample from which RNA was collected for microarrays, 2-3 ovaries per well were cultured with media changes every 24 hours for two days in the absence (controls) or presence (treated) of either Amh (human Anti-Mülerian hormone)(50ng/mL, R&D Systems Inc., USA), Fgf2 (rat Fibroblast growth factor 2)(50ng/mL, R&D Systems Inc., USA), Bmp4 (human Bone morphogenetic protein 4)(50ng/mL, R&D Systems Inc., USA), Gdnf (rat Glial derived neurotrophic factor)(50ng/mL, Calbiochem, USA), Fgf7 (human fibroblast growth factor 7/keratinocyte growth factor)(50ng/mL, R&D Systems Inc., USA), Kitlg (mouse Kit ligand)(50ng/mL, R&D Systems Inc., USA), Lif (rat leukemia inhibitory factor)(50ng/mL, Chemicon/Millipore, USA), Pdgf-ab (rat platelet derived growth factor AB heterodimer)(50ng/mL, R&D Systems Inc., USA), Tgfb1 (human transforming growth factor beta 1)(50ng/mL, R&D Systems Inc., USA). Two or three ovaries from the same culture well (from different rat pups out of the same litter) and receiving the same treatment were pooled and homogenized together. On any given day a culture experiment was performed, the treatment groups included untreated control ovaries and one to three different growth factor treatments.
Growth protocol
Four-day old female Sprague-Dawley rats (Harlan Laboratories, Inc., USA) were euthanized according to Washington State University IACUC approved protocols and the ovaries removed and cultured whole as described previously [Dole, G., et al., 2008].
Extracted molecule
total RNA
Extraction protocol
RNA was isolated from whole rat ovaries (2-3 ovaries per each sample) after homogenization in 1 ml Trizol™ reagent (Sigma-Aldritch, USA), according to manufacturer’s instructions.
Label
biotin
Label protocol
Biotin-labeled ssDNA were prepared according to the standard Affymetrix protocol from at least 300 ng total RNA (GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual, 2005-2209, Affymetrix).
Hybridization protocol
Following fragmentation, ssDNA were hybridized on Affymetrix Rat Gene 1.0 ST Array according to standard Affymetrix protocol. Chips were washed and stained in the Affymetrix Fluidics Station 4500.
Scan protocol
GeneChips were scanned using Affymetrix GeneChip® Scanner 3000.
Description
Gene expression data from rat P4 Ovary incubated in the presence of 50 ng/ml AMH for 48 hours
Data processing
The data were analyzed with Partek Genomic Suite 6.5 beta software (Partek Inc., St. Louis, MO) using RMA, GC-content adjusted algorithm background correction, quantile normalization, median polish methos for probesets summarization, and log values of probes signals using base 2. These VALUES were then unlogged for network analysis; the unlogged data are retained in the PRE-VALUE column below.
