Cell Culture and RNA purification Human breast cancer ZR-75.1 cells were propagated in DMEM medium supplemented with 10% FBS and antibiotics (100U/ml Penicillin, 100?g/ml Streptomycin and 250ng/ml Amphotericin-B). Before use, cells were plated at 20-40% confluence and maintained for 4 days in steroid-free medium (Phenol red-free DMEM medium, with 5% charcoal-stripped FBS and antibiotics), before stimulation with 5x10-8M 17?-estradiol (E2). At the indicated times, total cellular RNA was extracted from control (0, - E2) or E2–stimulated (+E2) cultures. 6 culture plates (150mm) were used for each time-point, and the experiment was repeated three times within two weeks. Poly(A)+ RNA was isolated from pools of 2mg total RNA, by batch-wise magnetic separation with Dynabeads (Oligo (dT)25; Dynal, Oslo, Norway) according to the manufacturer’s instructions, resuspended in diethyl pyrocarbonate-treated water, quantitated and diluted to a final concentration of 50ng/?l. Before further use, total and poly(A)+ RNAs were tested by agarose gel electrophoresis.
cDNA microarray probing cDNA microarray mRNA analysis was carried with UniGEM Human V 2.0 (Incyte Genomics, St. Louis, U.S.A.). 200ng of each poly(A)+ RNA derived from estrogen stimulated (+E2) cells were used to generate carbocyanine 5 (Cy5)-labeled cDNAs, that was mixed with common reference probes consisting of carbocyanine 3 (Cy3)-labeled cDNAs prepared from an equal amount of poly(A)+ RNAs purified from quiescent, hormone deprived cultures (-E2), for competitive hybridization to a cDNA microarray grid containing 9,182 human sequences of known genes and expressed sequence tags (representing 8,372 unique gene/ESTs clusters) and 192 internal controls (for reverse transcription, absolute sensitivity and differential expression QCs: human UniGEM V 2.0 screening services; Incyte Genomics, St. Louis, U.S.A.). cDNA synthesis, labeling and hybridization were generally repeated 3 times (in three cases 4 times), in separate experiments.
