Gc3000 7g scanner

Total RNA was annealed to an oligo‐d(T) primer with a T7 polymerase binding site. After generation of double‐stranded cDNA, copy RNA (cRNA) was transcribed which then formed the RNA template for a second round of amplification. At the end of this round, after synthesis of double‐stranded cDNA, biotin‐labelled cRNA was prepared using the Affymetrix Gene Chip (Affymetrix, Santa Clara, CA, USA) in vitro transcription labelling kit. Following clean‐up of the biotin‐labelled cRNA the material was assayed (Agilent Bioanalyser 2100) to ensure sufficient RNA of appropriate quality had been prepared. Labelled cRNA (12.5 μg) was fragmented and applied to HGU133 Plus 2.0 gene microarrays and hybridized over 16 h at 45°C in a rotating oven at 60 rpm. Post hybridization washing and sample staining was carried out using the Fluidics Station 400 and the Gene Chip Operating System. Gene chips were scanned using the GC3000 7G scanner and data processed for quality control using Expression Console software (Affymetrix) and further analysis was carried out using Qlucore Omics Explorer (Qlucore, Lund, Sweden).

The GeneChip® WT PLUS reagent kit (Affymetrix) was used to produce amplified, fragmented and labeled sense-strand DNA targets from total RNA. In brief RNA was reverse transcribed to produce single-stranded cDNA, double-stranded cDNA was then synthesized to produce a template for in vitro transcription, which generated complementary RNA (cRNA). cRNA was used to produce sense strand cDNA (ss-cDNA), which was purified and treated with RNase to hydrolyze the cRNA template. ss-cDNA was fragmented and labeled with biotin before hybridizing to GeneChip® Human Gene ST (GST) Arrays using the GeneChip® Hybridisation, Wash and Stain Kit. All samples were hybridized to arrays on the same day. Samples were then injected and hybridized onto GeneChip® Human GST cartridges and incubated in a GeneChip® hybridization oven 645 at 16 rpm for 16 h at 45 °C. Hybridization, washing and staining was performed using the Fluidics Station 400 and the Gene Chip Operating System (GCOS). The GC3000 7G scanner was used to scan gene chips and the expression console software (Affymetrix) was used to assess the quality of the data. Further analysis was carried out using Qlucore Omics Explorer (Qlucore, Lund, Sweden).
Data were normalized using the Robust Multi-array Average (RMA) and a principal component analysis was performed to determine the intensity distribution and eliminate sample outliers.