organ culture batch: 3 rna prep batch: 2 scan date: 2011-07-07 genotype: Sprague-Dawley rat gender: Female tissue: Rat ovary developmental stage: Rat ovary at day P0 incubated for 1 more day treatment: incubated in the presence of 50 ng/ml FGF2 for 24 hours
Treatment protocol
For each ovary sample from which RNA was collected for microarrays, 2-3 ovaries per well were cultured for 24 hours in the absence (controls) or presence (treated) of either Amh (human Anti-Mülerian hormone)(50ng/mL), CTGF (connective tissue growth factor )(500 ng/mL), progesterone (P4) (10E-6 M), estradiol (E2) (10E-6 M), TNF (tumor necrosis factor ) (1ng/mL), FGF2 (fibroblast growth factor 2) (50 ng/mL), Inhba (inhibin, beta A) (10 ng/mL). 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
Zero-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 P0 Ovary incubated in the presence of 50 ng/ml FGF2 for 24 hours
Data processing
The data were analyzed with Partek Genomic Suite 6.5 beta software (Partek Inc., St. Louis, MO) uing RMA, GC-content adjusted algorithm background correction, quantile normalization, median polish methos for probesets summarization, and log values of probes signals using base 2.
