Title: Affymetrix CEL analysis. Description:
nucleic acid labeling protocol
Labeling of samples was carried out according to the manufacturer’s standard protocol
Relative data are generated by the Affymetrix MAS5 algorithm with comparison Change expression ratio and comparison p-value
Relative data are generated by the Affymetrix MAS5 algorithm with Change expression and p-value
20µg of fragmented cRNA was hybridized to the Affymetrix HG_U133plus probe array cartridge in 200µl hybridization by incubation for 16hours at 45°C at constant rotation. The hybridized arrays were then washed and stained with Streptavidine Phycoerythrin and biotinylated anti-streptavidine antibody using and Affymetrix Fluidics station 450 following standard procedures.
(Parameters: Chamber type = Affymetrix- GeneChip Hyb Oven 640, Quantity of label target used = 20, Mass unit = Micro gram, time = 16, Tiny time unit = hours, Volume = 200, Volume unit = Micro litre, temperature = 45)
Total RNA was isolated from peripheral Blood Mononuclear cell using reagents and protocols provided in the RNeasy Mini Kit (Qiagen, Hilden, Germany). DNAse digestion was either performed including an on-column digestion step using DNase I as recommended by the manufacturer or using DNAfree reagents (Ambion, Austin, TX, USA) to remove genomic DNA contamination.
(Parameters: Extracted product = total_RNA, Amplification = none)
Peripheral blood (PB) samples were obtained before (day -2, -1 or 0) and after (median day 6, range 2-7) the first administrations of DAC (given at a dose of 15 mg/m2, 3 times daily on 3 consecutive days) from eight elderly patients with newly diagnosed AML treated within a phase II trial of DAC