Arcturus PicoPure RNA extraction kit was used for total RNA extraction according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 400 ng total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on MOUSE GeneChip Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol
GeneChips were scanned using the GeneChip® laser confocal fluorescence scanner.
Description
Gene expression data from embryos at E8.5.
Data processing
The data were analyzed with Affymetrix Microarray Suite Software. CEL files obtained by GeneChip Operating Software (GCOS, Affymetrix) were analyzed with the program dChip: a statistical method for model-based expression analysis (Li and Wong, 2001: "Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection." Proc Natl Acad Sci USA. 98, 31–36). After normalization, measures of the expression levels of each gene in each experiment were calculated using the perfect match/mismatch model described in Li and Wong, 2001: "Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application." Genome Biol. 2, 0032.1–0032.11 In parallel, CEL files obtained by GeneChip Operating Software (GCOS, Affymetrix) were analyzed with the program SAM. Data were normalized according to the GeneChip robust multiarray average (GCRMA) algorithm of the R/Bioconductor 1.4 package (http://www.bioconductor.org). Statistically significant expression changes were determined using permutation tests SAM (http://www-stat.stanford.edu/~tibs/SAM/). Genes regulated at least two fold between conditions were considered. The ∆ value was set to return a median false significant number = 0. The q values were 0 for the different gene lists created. Annotations were obtained through the Database for Annotation, Visualization, and Integrated Discovery (DAVID; http://david.niaid.nih.gov/david/beta/index.htm).