Isolated total RNA samples were processed as recommended by Affymetrix, Inc. (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetric, Inc., Santa Clara, CA). In brief, total RNA was initially isolated using TRIzol Reagent (Gibco BRL Life Technologies, Rockville, MD), and passed through an RNeasy spin column (Qiagen, Chatsworth, CA) for further clean up. Eluted total RNAs were quantified with a portion of the recovered total RNA adjusted to a final concentration of 1 ug/ul. All starting total RNA samples were quality assessed prior to beginning target preparation/processing steps by running out a small amount of each sample (typically 25-250 ng/well) onto a RNA Lab-On-A-Chip (Caliper Technologies Corp., Mountain View, CA) that was evaluated on an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).
Single-stranded, then double-stranded cDNA was synthesized from the poly(A)+ mRNA present in the isolated total RNA (10 ug total RNA starting material each sample reaction) using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen Corp., Carlsbad, CA ) and poly (T)-nucleotide primers that contained a sequence recognized by T7 RNA polymerase. A portion of the resulting ds cDNA was used as a template to generate biotin-tagged cRNA from an in vitro transcription reaction (IVT), using the BioArray High-Yield RNA Transcript Labeling Kit (T7) (Enzo Diagnostics, Inc., Farmingdale, NY). 15ug of the resulting biotin-tagged cRNA was fragmented to strands of 35-200 bases in length following prescribed protocols (Affymetrix GeneChip Expression Analysis Technical Manual).
Subsequently, 10 ug of this fragmented target cRNA was hybridized at 45°C with rotation for 16 hours (Affymetrix GeneChip Hybridization Oven 640) to probe sets present on an Affymetrix Mouse Genome 430 2.0 array.
The GeneChip arrays were washed and then stained (SAPE, streptavidin-phycoerythrin) on an Affymetrix Fluidics Station 450, followed by scanning on a GeneChip Scanner 3000. The results were quantified and analyzed using GCOS 1.1.1 software (Affymetrix, Inc.) using default values (Scaling, Target Signal Intensity = 500; Normalization, All Probe Sets; Parameters, all set at default values).
Get1, grainyhead epithelial transactivator 1. Also known as grainyhead-like 3 (Grhl3)