Isolated total RNA samples were processed as recommended by Affymetrix, Inc. (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetric, Inc., Santa Clara, CA). In brief, total RNA was initially isolated using TRI Reagent (Sigma, St. Louis, MO), and passed through an RNeasy spin column (Qiagen, Chatsworth, CA) for further clean up. All starting total RNA samples were quality assessed prior to beginning target preparation/processing steps by running out a small amount of each sample (typically 25-250 ng/well) onto a RNA Lab-On-A-Chip (Caliper Technologies Corp., Mountain View, CA) that was evaluated on an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).
Label
biotin
Label protocol
Single-stranded, then double-stranded cDNA was synthesized from the poly(A)+ mRNA present in the isolated total RNA (5 ug total RNA starting material each sample reaction) using the one cycle cDNA synthesis kit (Affymetrix, Inc.,Santa Clara CA ). A portion of the resulting ds cDNA was used as a template to generate biotin-tagged cRNA from an in vitro transcription reaction (IVT), using the Gene Chip IVT labeling kit.20ug of the resulting biotin-tagged cRNA was fragmented to strands of 35-200 bases in length following prescribed protocols (Affymetrix GeneChip Expression Analysis Technical Manual).
Hybridization protocol
Subsequently, 15 ug of this fragmented target cRNA was hybridized at 45°C with rotation for 16 hours (Affymetrix GeneChip Hybridization Oven 640) to probe sets present on an Affymetrix HU133 plus 2.0 array.
Scan protocol
The GeneChip arrays were washed and then stained (SAPE, streptavidin-phycoerythrin) on an Affymetrix Fluidics Station 450, followed by scanning on a GeneChip Scanner 3000.
Description
NME2
Data processing
The results were quantified and analyzed using GCOS 1.1.1 software (Affymetrix, Inc.) using default values (Scaling, Target Signal Intensity = 500; Normalization, All Probe Sets; Parameters, all set at default values).
