Two microarray experiments were performed. In Experiment 1, total RNA was isolated from individual whole embryos (4 vehicle control, 4 alcohol treated). Each embryo was immediately immersed in 700 ml TRIzol (Invitrogen) and homogenized using a Polytron. Extraction followed the TRIzol protocol. Ethanol precipitated RNA was resuspended in DEPC water. RNA was cleaned up using RNeasy mini-kit (Qiagen, Valencia, CA) The quality of RNA was assessed by the Agilent Bioanalyzer (Agilent Technologies, Waldbronn, Germany)and by spectrophotometry from 220 nm to 350 nm; concentration was determined from A260. Typical total RNA yields were 5-10 μg/embryo. Microarray analysis was performed at the Center for Medical Genomics at the Indiana University School of Medicine. Labeling and hybridization to Affymetrix Mouse Genome 430A GeneChips® (Affymetrix, Santa Clara, CA) were carried out following the manufacturer's suggested procedure. Fragmented biotinylated RNA from each embryo was separately hybridized to its own GeneChip for 17 hours at 42°C. The microarray analysis revealed striking differences among the 4 alcohol treated samples, which segregated as two separate pairs rather than one set of four; subsequently, it was noted that one
