RNA was extracted from all samples using TRIzol according to manufacturer's instructions (Invitrogen, Carlsbad, CA). Samples were then further purified using Qiagen miniprep RNA clean up (Qiagen, Valencia, CA) as per Affymetrix protocols for array preparation. Samples were quantified and assessed for quality using a Bioanalyzer 2100 instrument (Agilent Technologies, Palo Alto, CA).
Label
biotin
Label protocol
Five micrograms of purified RNA was reverse transcribed and used to make biotinylated cRNA according to standard Affymetrix protocol.
Hybridization protocol
Samples were hybridized to Canine Genome Version 2.0 Affymetrix oligonucleotide arrays according to manufacturer's instructions (Affymetrix, Santa Clara, CA) at the NCI Microarray Core Facility (Frederick, MD).
Scan protocol
Samples were scanned using an Affymetrix GeneChip scanner 3000. Data were collected using Affymetrix GCOS software.
Description
Spleen4
Data processing
Affymetrix .CEL files containing the raw, probe-level signal intensities were analyzed using Partek® software, version 6.4 (build 6.09.0310, Copyright 1993-2009, Partek Inc. Partek and all other Partek Inc. product or service names are registered trademarks or trademarks of Partek Inc., St. Louis, MO, USA.). Robust multichip averaging (RMA) was used for pre-processing of probe-level data (using only interrogating probes) including pre-background adjustment for GC content and probe sequence followed by RMA background correction. All chips underwent quantile normalization and probeset summaries were median polished (Bolstad, Irizarry, Astrand and Speed 2003).
