8 protocols
The log2-transformed data were quantile normalized
The bead level data (raw TIFF images and text files) obtained from BeadScan software were read using the Bioconductor beadarray package (version 2.2.0). The data were converted to bead-summary data using summarize function and the default logGreenChannelTransform transformation
Standard Illumina scanning protocol
Standard Illumina hybridisation protocol
Standard Illumina protocol
All W12 cells were derived through long-term in vitro passaging of cells from an explant culture of a low-grade squamous intraepithlial legion. The W12 squamous epithelial cells used were: W12Ser4p31, W12Ser4p86, W12Ser4B and G2p13.
RNA extraction
RNA was harvested using TRIzol (Life Technologies, Grand Island, NY, USA). QuantiTect reverse transcription kit (Qiagen, Crawley, UK) was used for cDNA synthesis, and quantitative PCR was performed using SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich, Dorset, UK). Expression levels were normalised against the mean of three house-keeping genes.
siRNA transfection
Cells were seeded into six-well plates at 2.4 x 105 cells per well (for protein extraction) or 12-well plates at 1x105 cells per well (for RNA extraction and growth curves) without antibiotics or feeder cells. Cells were transfected using 37nM of E7-141 siRNA or non-targeting control (NTC) siRNA.