Total RNA was isolated using TRIzol Reagent (Invitrogen) followed by column based clean up (RNeasy, Qiagen).
Label
biotin labeled
Label protocol
The amplification and labeling of the RNA samples were carried out according to the manufacturer's instructions (Affymetrix, Santa Clara, CA). Briefly, total RNA was quantified by UV-spectroscopy and its quality evaluated by analysis on a LabChip (BioAnalyzer, AGILENT Technologies, Santa Clara, CA). In the presence of an oligo(dT)24 primer containing a T7 RNA polymerase promoter (Operon Biotechnology, Huntsville, AL, USA) five micrograms of total RNA were used for double-stranded cDNA synthesis (SuperScript transcriptase, Life Technologies, Inc., Carlsbad, CA). Labeled complementary RNA (cRNA) was prepared from double-stranded cDNA by in vitro transcription using the bioarray high yield RNA transcription kit (Enzo Diagnostics, Farmindale, N.Y., USA). After cleanup (Qiagen, Hilden, Germany), the biotin-labeled cRNA was fragmented by alkaline treatment (40 mM Tris-acetate (pH 8.2), 100 mM potassium acetate, and 50 mM magnesium acetate) at 94°C for 35 minutes.
Hybridization protocol
Fifteen micrograms of each cRNA sample was hybridized for 16 hours at 45°C to human expression Affymetrix HG U95Av2 GeneChip arrays. Following the hybridization arrays were washed and stained with streptavidin-phycoerythrin using a fluidics station in accordance with the Affymetrix protocol.
