strain: Sprague-Dawley gender: Female tissue: ovary developmental stage: day P4 cultured for 2 more days
Treatment protocol
Ovaries dissected from 4-day old female rat pups were maintained in a whole organ culture system on floating filters (0.4 µm Millicell-CM; Millipore Corp., Billerica, MA, USA) in 0.5 ml DMEM-Ham's F-12 medium (1:1, vol/vol; Life Technologies, Inc.) containing 0.1% BSA (Sigma), 0.1% albumax (Life Technologies, Inc.), 0.05 mg/ml L-ascorbic acid (Sigma), and 27.5 µg/ml transferrin (Sigma) in a 4-well culture plate (Nunc plate; Applied Scientific, South San Francisco, CA, USA). Medium was supplemented with final concentration 5 µg/ml gentamicin, 3.25 µg/ml streptomycin, and 3.25 units/ml penicillin to prevent bacterial contamination. Ovaries were treated with no factor (control) or NT3 (rh NT3, 50 ng/ml; R&D Systems, Minneapolis, MN, USA).
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
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 cRNA were prepared according to the standard Affymetrix protocol from at least 3 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Following fragmentation, cRNA were hybridized on Affymetrix GeneChip Rat Genome 230 2.0 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 cultured for 48 hours with NT3
Data processing
The data were analyzed with GeneSpring GX7 (Agilent Technologies, Santa Clara, CA) software using MAS5.0 algorithm for pre-processing and global scaling as normalization method. The trimmed mean target intensity of each array was set to 210.
