normalization data transformation protocol
Samples were processed using methods from the lumi package in R. Background correction using the bgAdjust method, variance stabilization and quantile normalization were performed on the raw data. All pre-processing methods use lumiExpresso to combine multiple steps and a log2 transform in also performed.
array scanning protocol
Microarrays are scanned using the Illumina BeadArray Reader which runs BeadScan Software. Illumina recommended protocols are used. (Parameters: Scanning hardware = OTHER: BeadArray Reader [Illumina], Scanning software = Scanning software)
The samples were obtained from the FHL-124 cell line, that is a non-virally transformed cell line generated from human capsule-epithelial explants. FHL-124 cells were routinely cultured at 35C in a humidified atmosphere of 95% air and 5% CO2, in Eagle's Minimal Essential Medium (EMEM) (Gibco, Paisley, UK) supplemented with 5% v/v foetal calf serum (Gibco) and 50ug/ml gentamicin for 48 hours. Medium was then removed and replaced with Eagle's Minimal Essential Medium (EMEM) supplemented with 50ug/ml gentamicin for 24 hours. Following this period, cells were either maintained in the same medium or exposed to 1 or 2uM sulforaphane (Sigma, Poole, Dorset) for a further 24 hours. At end-point, samples were extracted from the dishes and snap-frozen and stored in a -80C freezer prior to extracting RNA using the RNeasy mini kit (Qiagen Ltd, Crawley, UK).
nucleic acid extraction protocol
Total RNA was extracted using Qiagen MicroRNA extraction kits following the manufacturers protocols. Amplificiation was performed using Illumina TotalPrep-96 RNA Amplification Kit. On column DNA digestion was performed using a RNase-free DNase I kit (Qiagen). RNA samples were frozen at -80C until required. (Parameters: Extracted product = total_RNA, Amplification = RNA polymerases)
Cell lines were routinely cultured at 35C in a humidified atmosphere of 95% air and 5% CO2, in Eagle's Minimal Essential Medium (EMEM) (Gibco, Paisley, UK) supplemented with 5% v/v foetal calf serum (Gibco) and 50ug/ml gentamicin. (Parameters: start time = 24, time unit = hours, min temperature = 35, temperature unit = C, media = Eagle's Minimal Essential Medium)