tissue: colorectal cancer disease state: Lynch syndrome age: 48 gender: male
Extracted molecule
total RNA
Extraction protocol
Three to five 5 μm sections per FFPE block were used for RNA isolation. RNA deparaffinization, extraction and purification were performed using the High Pure RNA Paraffin Kit (Roche, Castle Hill, Australia) according to the manufacturer's instructions. RNA concentration was determined using a NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE), and samples yielding sufficient RNA (200 ng) with 260/280 ratios >1.8 were selected.
Label
Cy3
Label protocol
Ligated products were PCR-amplified and labeled with a universal Cy3-coupled primer, after which single-stranded labeled products were precipitated and then hybridized to WG gene expression BeadChips as previously described (Kuhn et al. 2004).
Hybridization protocol
Standard Illumina hybridization protocol.
Scan protocol
Standard Illumina iScan N240 scanning protocol.
Description
AH37
Data processing
Illumina gene expression data were loaded and interpreted in GenomeStudio software (Illumina, San Diego, CA). Data were normalized using background correction, the cubic spline method and plate scaling. Normalized gene expression data were then exported, and only RefSeq features with a detection P value of ≤0.01 in at least 80% of the samples (n=122) were used in further analyses. Technical replicates within samples were average combined to create one signature per tumor. Next, data were loaded into MeV v4 (Saeed et al. 2003), where they were log2 transformed and median centered across assays. Significance analysis of microarrays (SAM) was used to identify significantly differentially expressed genes (false-discovery rate, FDR ≤ 5%). Hierarchical clustering and SAM were done in MeV (Saeed et al. 2003).
