Biotinylated complimentary RNA (cRNA) was prepared from 10 ug of total RNA as per the Affymetrix GeneChip Technical Analysis Manual (Affymetrix, Santa Clara, CA). Double-stranded cDNA was synthesized using SuperScriptII (Invitrogen, Carlsbad, CA) and oligo(dT)24 primers. Biotin-labeled cRNA was prepared by cDNA in vitro transcription using the BioArray High-Yield RNA Transcript Labeling kit (Enzo Biochem, New York) incorporating biotinylated UTP and CTP.
Hybridization protocol
10 μg of labeled cRNA was hybridized to Affymetrix Bovine Genome arrays for 16 hours at 45°C as described in the Affymetrix Technical Analysis Manual (Affymetrix, Santa Clara, CA).
Scan protocol
GeneChips were stained with Streptavidin-Phycoerythrin, followed by an antibody solution and a second Streptavidin-Phycoerythrin solution, with all liquid handling performed by a GeneChip Fluidics Station 400. GeneChips were scanned with the Affymetrix GeneChip Scanner 3000 (Affymetrix, Santa Clara, CA).
Description
rumen papillae from third week of grain challenge
Data processing
Gene level data was generated using the GC Robust Multichip Analysis (GCRMA) preprocessor in GeneSpring GX 10 (Agilent Technologies)
