cell type: FFPE breast cancer breast cancer subtype: Normal
Extracted molecule
total RNA
Extraction protocol
For primary tumours and normal breast tissue, 10μm thick sections were cut from FFPE tissue blocks. The sections were dewaxed in xylene, placed through 100% alcohol and allowed to dry. The samples were needle microdissected to ensure the proportion of tumour (or normal epithelium) was greater than 80%, prior to placement into lysis buffer (Agencourt Formapure kit, Beckman Coulter, Beverly, MA, USA). Tissue was digested as per kit protocol (incubate at 70oC for 1 hr, then add 20 µl of Proteinase K and incubate at 55oC for 1hr). Total RNA was extracted via a standard TRIZOL(Sigma)/chloroform protocol. For cell lines, total RNA was extracted using the total RNA protocol from the mirVana miRNA Isolation Kit (Ambion, TX, USA). All samples underwent DNase treatment with the Ambion DNA-free kit (Ambion, TX, USA).
Label
biotin
Label protocol
Biotinylated cDNA were prepared from 0.2-1ug total RNA using the high-throughput Gene expression profiling, the DASL Assay (cDNA-mediated annealing, selection, extension and ligation)
Hybridization protocol
Fluorescently labelled cDNA PCR products were hybridized to the Illumina Sentrix BeadChip U1536-16 according to the protocols provided by the manufacturer.
Scan protocol
Arrays were scanned using the Illumina Bead Array Reader Confocal Scanner
Description
4687786009_E
Data processing
The BeadChips were scanned with the Illumina iScan Reader. Data were imported into GenomeStudio (Illumina), from which raw data with background subtraction were exported to PARTEK Genomics Suite (St. Louis, Missouri, USA) for further analysis. Probes with a maximum intensity value of less than 150 units across all samples were excluded. Of the 1145 probes present on the array, 1037 were used for subsequent analyses. Raw probe intensities were shifted, such that the minimum probe intensity for each sample was equal to 1. All values were transformed by taking logs (base 2), followed by quantile normalisation.
