RNA was extracted from frozen tissue samples by homogenization in trizol followed by chloroform separation and extraction in an RNAeasy mini column (Qiagen Inc., Germantown, MD) prior to cleaning using the RNase-Free DNaseSet (Qiagen Inc., Germantown, MD). Microarray was performed using the GeneChip WT PLUS Reagent Kit (Affymetrix, Santa Clara, CA) to ampligy 250 g of RNA for each sample. All RNA used for microarray had a minimum RNA Integrity Number (RIN) value of 6.5.
Label
biotin
Label protocol
not provided
Hybridization protocol
not provided
Scan protocol
not provided
Data processing
Affymetrix (Santa Clara, CA) HTA 2.0 arrays were processed using the 'oligo'(14) BioConductor package, quality-controlled with array QualityMetrics(15). After exclusion of clear outliers, the remaining arrays were processed using the TAC4.0 software from Affymetrix/Thermo Fisher using their RMA-SST algorithm and imported back into this workflow. Non-coding and non-annotated genes were removed from the dataset; this strategy identified 25,049 of 70,523 probes for analysis. These remaining probes were collapsed by taking the probeset for each gene with the maximum value for all samples; this strategy identified 23,327 genes which were included in the analysis represented here. Differentially expressed genes (as defined by a Benjamini-Hochberg FDR adjusted p-value of less than 0.05) were identified using limma(16) using a paired design.
