Frozen mammary gland tissue, collected from four to five mice at day seven (placebo) or week 4 (estradiol), was pooled and RNA was prepared using Trizol reagent and the RNeasy clean up protocol.
Data processing
Five hundred ng of total RNA was amplified and labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit according to the manufacturer's protocol. For each two color comparison, 750 ng of each Cy3- and Cy5-labeled cRNA were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 hours in a rotating hybridization oven according to the Agilent 60-mer oligo microarray processing protocol prior to washing and scanning with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software (v8.1), using default for all parameters.
