Fifty micrograms of RNA from each mouse liver sample were enriched for poly A+ RNA using the AtlasTM Pure Total RNA Labeling system (Clontech, Palo Alto, CA), and reverse transcribed in the presence of [33P]dATP, dCTP, dGTP, dTTP and AMV reverse transcriptase to produce cDNA probes. The probes were purified using the ProbeQuant™ G-50 Micro Columns (Amersham Pharmacia Biotech Inc., Piscataway, NJ), and then denatured in the presence of Cot-1 DNA. The Clontech AtlasTM Mouse 1.2 Arrays (7853-1)(Palo Alto, CA) were prehybridized in 5 ml of ExpressHyb (Clontech) and 0.5 mg of heat denatured sheared salmon sperm for 30 minutes at 68C prior to probe incubation. The probes were added and incubated overnight at 68C degrees before washing four times for 30 minutes each time with 2X SSC, 1% SDS, then once with 0.1X SSC, 0.5% SDS for 30 minutes, and once with 2X SSC for 5 minutes at 68oC. Blots were visualized by placing them on a K-screen for 3-4 days (Bio-Rad, Hercules, CA). Arrays were visualized from the K-screen, and analyzed and quantified with a phosphoimager (Molecular Imager FX, Bio-Rad, Hercules, CA) and the ResGen PathwaysTM Universal Microarray Analysis SoftwareTM (Research Genetics, Carlsbad, CA). Local background was subtracted automatically from the individual spot intensities, and spot intensities were normalized to the mean intensity of all spots on the array. This is called datapoint normalization on the ResGen Pathways Microarray Analysis SoftwareTM. Normalized gene intensities on the arrays probed with 4-NP treated mouse livers and untreated mouse liver cDNA were compared, and fold differences were calculated. Keywords = 4-nonylphenol
