Genome wide expression analysis by application of the Human Gene 2.0 ST array (Affymetrix) and relative quantification of transcripts by real-time RT-PCR

Total RNA was extracted from 1x10⁷ cells induced for 24 h with 1 μM ß-estradiol using the Qiagen RNeasy Mini Kit. Expression analysis starting from 100 ng of total cellular RNA was performed using the Ambion WT Expression Kit (Applied Biosystems) and subsequently the GeneChip WT Terminal Labeling and Hybridization Kit (Affymetrix) followed by the GeneChip Human Gene 2.0 ST array (Affymetrix) according to the manufacturer's protocol. All affymetrix CEL files have been processed in Bioconductor/R using robust multiarray average (RMA) for normalization and summarization and limma for differential expression and significance. Quality has been checked using the array QualityMetrics package. Additional filtering based on the fold change between the two conditions was applied with different stringency, individually described in the legend of the tables and figures. Analyzation and visualization of the Microarray was performed using Genesis, available at http://genome.tugraz.at. Quantitative RT-PCR analysis was performed as described previously [72]. Primers used for RT-qPCR were designed applying Primer3 software (http://primer3.ut.ee/) and selection of mature transcripts was ensured by amplification across exon-exon junctions. Primers used for quantitative RT-PCR are summarized in S3 Table. All data were normalized for the relative abundance of the Actin B transcript.