Total RNA was isolated using RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions.
Label protocol
fluorescently-labelled cRNA was generated by in vitro transcription using Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Palo Alto, CA) according to the manufacturer’s instructions.
Universal Human Reference RNA (Stratagene, Cedar Creek, TX), used as reference control in all microarray gene-profiling experiments, consisted of equal amount of total RNA from 10 human cancer cell lines.
Label protocol
fluorescently-labelled cRNA was generated by in vitro transcription using Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Palo Alto, CA) according to the manufacturer’s instructions.
Hybridization protocol
cRNA products were purified using RNeasy columns (Qiagen). Samples had to contain 10-20 picomoles of cyanine dye/ug of cRNA to be considered suitable for subsequent hybridization. 1 ug of Cy5-labelled cRNA was mixed with the same amount of Cy3-labelled reference cRNA and, then, mixed cRNAs were fragmented to an average size of 50-100 nt by incubation at 60°C for 30 min using in situ Hybridization kit-plus (Agilent). Samples were hybridised on Agilent Human 1A Oligo Microarray (V2), ink-jet printed microarray, comprising 20,173 (60-mer) experimentally validated oligonucleotide probes (features). After hybridization for 17 h at 60°C, slides were washed according to Agilent SSPE protocol instructions and then scanned using a confocal laser scanner (Agilent Technologies).
Scan protocol
Fluorescence data were analysed with Feature Extraction Software v7.5 (Agilent Technologies).
