RNA was isolated using the PicoPure RNA Isolation kit (Arcturus Engineering, Inc. Mountain View, CA) digested with Dnase.
Label
biotin
Label protocol
Two rounds of linear amplification were performed by combining reagents supplied in MessageAmp™ II-Biotin Enhanced Single Round aRNA Amplification Kit (Ambion, Catalog# AM1791, Austin, TX) and MessageAmp™ II aRNA Amplification Kit (Ambion, Catalog# AM1751), resulting in biotin-labeled antisense cRNA [21].
Hybridization protocol
Per sample,15μg of biotin-labeled cRNA sample was fragmented and processed for hybridization to Human Genome U133A 2.0 Genechips (Affymetrix Inc., Santa Clara, CA), according to the Expression Kit User Manual (http://www.affymetrix.com/support/technical/manuals.affx)).
Scan protocol
Arrays were washed and stained using the Midi_euk2v3_450 protocol (V4). Fluorescent intensity emitted by the labeled target was measured using a GeneChip Scanner 7G (Affymetrix Inc.).
