Animals. Virgin Lewis rats were from Charles River Laboratories (Wilmington, MA), housed in a temperature-controlled room with 12-h light/dark schedule, fed food (Teklad 8640; Teklad, Madison, WI), and water ad libitum. All the procedures followed University of California Animal Care and Use Committee guidelines.
Estradiol Treatment. All doses of E2 were packed in individual silastic capsules (size 0.078 inch i.d. x 0.125 inch o.d., 2 cm in length; Dow Corning Corporation, Midland, MI) in a cellulose matrix. Control animals received silastic capsules containing only cellulose. All silastic capsules were dorsally implanted s.c., and primed before implantation by soaking in media 199 (GIBCO) overnight at 37°C.
Persistent Effect of Different Doses of Estradiol on Gene Expressions in the Mammary Gland. At 9 week old rats were divided into 3 groups, each consisting of 3 rats, and receiving one of the following treatments: (i) Control, (ii) 10 ^g E2 (non-protective), and (iii) 200 |ig E2 (protective). Each treatment was continued for 3 weeks. At the end of the treatment, the silastic capsules were removed. The rats were terminated, 8 weeks after the removal of the hormone treatment. Mammary glands were removed, immediately frozen in liquid N, and stored at -80°C.
RNA Isolation. RNA was isolated from frozen samples using Trizol (Life Tech). Total RNA was subjected to DNAse treatment, and further purified with RNeasy columns (Qiagen). The quality of RNA was analyzed using the Agilent 2100 Bioanalyzer, and quantified using a Hitachi UV spectrophotometer.
Microarray Analysis. Using Agilent Rat cDNA Microarrays, with 14,815 unique clones, we analyzed genes from Rattus norvegicus and Rattus rattus which are annotated as "mRNA" or "gene EST" in GenBank. Experiments were performed in replicates. Samples were labeled and reverse labeled with Cy3 and Cy5. The combined plots show high reproducibility of the experimental set. Bioinformatic analysis was carried out using Rosetta Resolver software.
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