Messenger RNA is amplified and labeled from 2 mg of total RNA in two steps. In the first step, mRNA is converted to double-stranded cDNA using Superscript Reverse Transcriptase (Invitrogen) and an oligo-dT primer linked to a T7 RNA polymerase binding site sequence (Affymetrix). In the second step, amplified and labeled cRNA (the target) is produced in an in vitro transcription reaction using T7 RNA polymerase and biotin-CTP (Affymetrix.). Following removal of free nucleotides, target yield is measured by UV260 absorbance.
Hybridization protocol
Labeled target is fragmented at 95o C in the presence of high magnesium concentration. The fragmented material is combined with biotinylated hybridization control oligomer and biotinylated control cRNAs for BioB, BioC, BioD and CreX (Affymetrix) in hybridization buffer. Ten g of target is hybridized with the GeneChip (insert array name) array (Affymetrix) overnight, followed by washing, staining with streptavidin-phycoerythrin (Molecular Probes), signal amplification with biotinylated anti-streptavidin antibody (Vector Labs), and a final staining step on the Fluidics Station 450 (Affymetrix). The distribution of fluorescent material on the processed array is determined using the Affymetrix 3000 GeneArray laser scanner with the 7G upgrade.
Scan protocol
Image inspection is performed manually immediately following each scan. All array scanning and data processing on the Affymetrix system is performed with GeneChip Operating System (GCOS) software.
Data processing
The probe-level Cel file is processed with Affymetrix Microarray Suite, version 5.0 (MAS 5.0) software. The GeneChip expression arrays contain control probe sets for both spiked and endogenous RNA transcripts (e.g., BioB, BioC, BioD, CreX and species-specific actin and GAPDH). Following image processing and absolute analysis of the array pattern with MAS, six values are examined to assess overall assay performance: background, noise, average Signal, % Present, ratio of Signal values for probe sets representing the 5’ and 3’ ends of actin and GAPDH transcripts, and total Signal for probe sets for BioB, BioC, BioD and CreX. Assays demonstrating poor or marginal performance are flagged. Using MAS 5.0, array data is globally scaled to a uniform, average target intensity for all assays prior to further analysis.
