U87 human glioma cells were cultured in DMEM with 10% FBS, antibiotics and L-glutamine. Fertilised chicken eggs were kept and prepared as described in (Hagedorn, M et al. 2002. PMID: 12460903)
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using Qiagen (Valencia, CA) RNeasy columns
Label
Cy3
Label protocol
RNA was reverse transcribed (cDNA Synthesis System Kit, Roche Diagnostics), purified and transcribed in the presence of Cy3 and Cy5 labels (Megascript D7 Kit, Ambion, Austin, TX).
Hybridization protocol
RNA was hybridised to Agilent Chicken (V1) microarrays and Human GE v2 microarrays (Agilent Technologies, CA). Due to the proximity of tumour cells to the surrounding chicken cells no attempt was made to separate them, instead pooled RNA from both types of cell were hybridised to chicken and human arrays. CAM cells distant from the tumour were hybridised to chicken arrays.
Scan protocol
Scanning and spot analysis were done according to the manufacturer’s instructions.
Data processing
Controls were removed from the data after scanning. The arrays were log2 transformed and normalised using between arrays quantile normalisation in the R software package preprocessCore from BioConductor
