Amersham hybridization solution

Ten micrograms of biotin-labeled aRNA was fragmented using 5 µl of fragmentation buffer in a final volume of 20 µl, then was mixed with 240 µl of Amersham hybridization solution (GE Healthcare Europe GmbH) and injected onto CodeLink Uniset Rat Whole Genome bioarrays (40K) containing 36,000 rat oligonucleotide gene probes (both from GE Healthcare Europe GmbH). Arrays were hybridized overnight at 37°C at 300 rpm in an incubator. The slides were washed in stringent TNT buffer (0.1 M Tris-HCl, 0.15 M NaCl, 0.05% Tween-20) at 46°C for 1 h, and then a Streptavidin-Cy5 (GE Healthcare) detection step was performed. Each slide was incubated for 30 min in 3.4 ml of Streptavidin-Cy5 solution as described previously (Fevre-Montange et al., 2006 (link)), then was washed four times in 240 ml of TNT buffer, rinsed twice in 240 ml of water containing 0.2% Triton X-100, and dried by centrifugation at 50 g.
The slides were scanned using a Genepix 4000B scanner (Axon) and GenePix software (Molecular Devices), with the laser set at 635 mm, the laser power at 100%, and the photomultiplier tube voltage at 60%. The scanned image files were analysed using CodeLink expression software, version 4.2 (GE Healthcare), which produces both a raw and normalized hybridization signal for each spot on the array.

Ten micrograms of biotin-labeled aRNA was fragmented using 5 μl of fragmentation buffer in a final volume of 20 μl and was then mixed with 240 μl of Amersham hybridization solution (GE Healthcare Europe GmbH, Freiburg, Germany) and injected onto CodeLink Uniset Human Whole Genome bioarrays containing 5,5000 human oligonucleotide gene probes (GE Healthcare Europe GmbH, Freiburg, Germany) as described previously (11 (link)). Arrays were hybridized overnight at 37°C at 300 rpm in an incubator. The slides were washed in stringent TNT buffer at 46°C for 1 h, then a streptavidin-cy5 (GE Healthcare) detection step was performed. Each slide was incubated for 30 min in 3.4 ml of streptavidin-cy5 solution, was then washed four times in 240 ml of TNT buffer, rinsed twice in 240 ml of water containing 0.2%Triton X-100, and dried by centrifugation at 600 rpm. The slides were scanned using a Genepix 4000B scanner (Axon, Union City, USA) and Genepix software, with the laser set at 635 mm, the laser power at 100%, and the photomultiplier tube voltage at 60%. The scanned image files were analyzed using CodeLink expression software, version 4.0 (GE Healthcare), which produces both a raw and normalized hybridization signal for each spot on the array. Transcriptomic data have been deposited in Gene Expression Omnibus under the accession number GSE120350.