RNA was isolated from live cells using the Qiagen RNeasy® Plus Mini Kit following the manufacturer's instructions (Qiagen, Valencia, CA).
Label
Biotin
Label protocol
Oligonucleotide microarray analysis of RNA expression levels was performed in the Vermont Genetics Network Microarray Facility using the Affymetrix GeneChip Platform (Affymetrix Inc., Santa Clara, CA) according to manufacturer's protocols. In brief, the Nugen Ovation system v.2 with SPIA® RNA amplification was employed to convert 50ng of total RNA to cDNA. This isothermal RNA amplification system produces 5-12 ug of anti-sense cDNA targets that is followed by several steps to produce sense strand cDNA to be fragmented, biotinylated and hybridized to the GeneChip.
Hybridization protocol
After purification and fragmentation, biotinylated-cDNA targets were hybridized to the Mouse Gene 1.0 ST Arrays oligonucleotide arrays for 16 hours at 45oC. Hybridized arrays were washed and stained with streptavidin-phycoerythrin followed by sequential incubations with biotin-coupled polyclonal anti-streptavidin antibody and streptavidin phycoerythrin as a fluorescent amplification step.
Scan protocol
After staining, arrays were scanned (3000-7G Scanner, Affymetrix Inc., Santa Clara, CA) and data collected for statistical analysis.
