Total RNA was extracted from snap-frozen tissues using the PerfectPure RNA Tissue kit with on-column Dnase I treatment (5-PRIME). Extracted total RNA was then quality verified by Agilent Bioanalyzer
Label
biotin
Label protocol
Four ug of SPIA amplified DNA was then used to generate ST-cDNA using the WT-Ovation Exon Module v1 (NuGEN Technologies, Cat #2000) and again cleaned up with the Qiagen column as above. Five micrograms of this product was fragmented (average fragment size = 85 bases) and biotin labeled using the NuGEN FL-Ovation cDNA Biotin Module, v2 (NuGEN Technologies, Cat. #4200) per the manufacturer's recommended protocol.
Hybridization protocol
Biotin-labeled cDNA was mixed with Affymetrix eukaryotic hybridization buffer (Affymetrix, Inc., Santa Clara, CA), placed onto Affymetrix Mouse Gene 1.0 ST Arrays [probe set (exon) version], and incubated at 45C for 18 h with 60 rpm rotation in an Affymetrix Model 640 Genechip Hybridization Oven. Following hybridization, the arrays will be washed, stained with streptavidin-phycoerythrin (Molecular Probes, Inc., Eugene, OR), signal amplified with antistreptavidin antibody (Vector Laboratories, Inc., Burlingame, CA) using the Affymetrix Model 450 Fluidics Station.
Scan protocol
Arrays were scanned with the Affymetrix Model 3000 scanner with 7G upgrade and data collected using the using the GeneChip operating software (GCOS) v1.4.
Description
keratoachanthoma from a K14-Cre;Zmiz1 double transgenic mouse
Data processing
Data were processed using Partek Genomics Suite software. Corrected intensities were RMA normalized. probe group file: MoEx-1_0-st-v1.r2 meta-probeset file: MoEx-1_0-st-v1.na27.mm9.transcript
