Mix of equal amount of total RNAs from our cohort was used as reference control in all microarray gene-profiling experiments. Amplified reference cRNA was labelled with cyanine 3-CTP (Perkin-Elmer, NEN Life Science, Boston, MA, USA) in each experiment.
For all 29 specimens, fluorescently-labelled cRNA was generated by in vitro transcription using Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Palo alto, CA, USA) according to the manufacturer’s instructions. Amplified cRNA of each patient was labelled with cyanine 5-CTP (Perkin-Elmer, NEN Life Science, Boston, MA, USA) in each experiment. Mix of equal amount of total RNAs from our cohort was used as reference control in all microarray gene-profiling experiments. Amplified reference cRNA was labelled with cyanine 3-CTP (Perkin-Elmer, NEN Life Science, Boston, MA, USA) in each experiment. Moreover, cRNA products were purified using RNeasy columns (Qiagen). 1 g 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 hybridized on Agilent Human 1A Oligo Microarray, ink-jet printed microarray, comprising 20,173 (60-mer) experimentally validated oligonucleotide probes (features).33 After hybridization for 17 h at 60°C, slides were washed according to Agilent SSC protocol instructions and then scanned using a confocal laser scanner (Agilent Technologies).
Data processing
linear and lowess normalization (Agilent settings)
