Strain: C57BL/6J Gender: Male Age: 10 weeks Tissue: intestinal mucosa
Extracted molecule
total RNA
Label
R-phycoerythrin
Description
Eight micrograms of total RNA were converted into double-stranded cDNA (ds-cDNA) by use of the SuperScript double-stranded cDNA synthesis kit and GeneChip T7-oligo(dT) promoter primer kit. After second-strand synthesis, the reaction mixture was purified using the GeneChip sample cleanup module. In vitro transcription was performed on the ds-cDNA with the Enzo RNA transcript labeling kit. Biotinylated cRNA was purified using the GeneChip sample cleanup module and fragmented by incubating 94°C for 35 min. The biotinylated cRNA (final 0.05 mg/ ml) was hybridized with Mouse Expression Array 430A for 16 h at 45°C. After stringent washing in a microfluidics station 400, cRNA was stained with R-phycoerythrin streptavidine and scanned after antibody amplification. Fluorescence intensities were measured with an Affymetrix Scanner. Image output files were inspected for hybridization artifacts, and based on the fluorescence intensity differences between perfect match and mismatch probes, a hybridization intensity was calculated for each transcript using Microarray Suite 5.0. Default parameters optimized by the manufacturer for this microarray chip were used for all signal analysis.
Data processing
Normalized by the value of acidic ribosomal phosphoprotein PO (1419441_at)
