An aliquot of 20-30 x106 leukemic bone marrow cells was used to prepare total RNA applying the Trizol (Invitrogen) purification method according to the manufacturers’ instructions. Total RNA was retro-transcribed and labeled using a MICROMAX TSA Labeling Kit (PerkinElmer). Two ug of total RNA were used in each reaction but only half of the labeled cDNA was actually hybridized to the microarrays. Microarray hybridisation was carried out in a dual slide chamber (HybChamber, Gene Machines, San Carlos, CA, USA) humidified with 100 µl of 3 x SSC. Labeled cDNA was dissolved in 40 µl of hybridisation buffer, denatured at 90°C for 2 min in a thermal cycler and applied directly to the slides. Microarrays were covered with 22 x 40 mm cover slip and hybridized overnight at 65°C by immersion in a high precision water bath (W28, Grant, Cambridge, UK). Post-hybridization washing was performed according to the MICROMAX TSA Detection kit (PerkinElmer). . Digital images were generated in a GSI Lumonics LITE dual confocal laser scanner (ScanArray Microarray Analysis Software) and processed with QuantArray Analysis Software (GSI Lumonics, Ottawa, Canada). Normalized data were determined using the publicly available software MIDAS (TIGR Microarray Data Analysis System: http://www.tigr.org/softlab/). Normalized values (lowess normalization) were calculated for each spot, and converted in logarithmic scale. Final values correspond to log(2) ratio of the normalized intensities.
Keywords = human bone marrow Keywords = childhood leukemia Keywords = gene expression profiling
