RNA sample preparation, microarray hybridization, and expression analysis.
RNA preparation and array hybridizations were performed at Rosetta Inpharmatics (Seattle, Washington, United States). The custom ink-jet microarrays used in this study (Agilent Technologies, previously described [21,34]) contain 2,186 control probes and 23,574 noncontrol oligonucleotides extracted from mouse UniGene clusters and combined with RefSeq sequences and RIKEN full-length clones.
Mouse livers were homogenized and total RNA extracted using TRIzol reagent (Invitrogen, Carlsbad, California, United States) 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 microarray slides 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). Gene expression is reported as the mlratio relative to the pool derived from 150 mice randomly selected from the F2 population. For subsequent analyses, mlratio data are assumed to be normally distributed, a valid assumption as previously demonstrated [21,30]. The error model used to assess whether a given gene is significantly differentially expressed in a single sample relative to a pool comprised of a randomly selected subset of 150 samples has been extensively described and tested in a number of publications [35,36].