Liver samples extracted from the 427 Caucasian individuals were homogenized, and total RNA extracted using TRIzol reagent (Invitrogen) according to manufacturer's protocol. Three micrograms of total RNA was reverse transcribed and labeled with either Cy3 or Cy5 fluorochrome. Purified Cy3 or Cy5 complementary RNA was hybridized to at least two single microarrays with fluor reversal for 24 h in a hybridization chamber, washed, and scanned using a laser confocal scanner. Arrays were quantified on the basis of spot intensity relative to background, adjusted for experimental variation between arrays using average intensity over multiple channels, and fitted to an error model to determine significance (type I error), as previously described [42]. Gene expression is reported as the mean-log ratio relative to the pool derived from 192 liver samples selected for sex balance from the Vanderbilt and Pittsburgh samples, because the RNA from the Merck samples had been amplified at an earlier date. The error model used to assess whether a given gene is significantly differentially expressed in a single sample relative to a pool composed of a randomly selected subset of samples has been extensively described and tested in a number of publications [42–44].
