Total RNA was extracted from U-OFF or U-2OSbeta cells treated with ethanol vehicle for 6 hours using the RNAqueous Total RNA Isolation Kit. RNA was treated with DNase using the DNA-Free DNase Treatment and Removal Reagents prior to use with the microarray.
Data processing
Gene expression analysis was conducted using Agilent Human1Av2 arrays. Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng of total RNA, Cy3 or Cy5 labeled cRNA was produced. For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software (v7.5), using defaults for all parameters.
