Miniarray is a diagnostic platform to recognize different subtypes of ALL. It was constructed arraying on glass slides PCR-amplified cDNAs of 30 genes best discriminating ALL-B and ALL-T patients, 10 tags that are differentially expressed in all leukemia samples and 9 housekeeping genes used as hybridization controls. PCR products are printed in quadruplicate in each miniarray and each slide contains four miniarrays. cDNA inserts were PCR amplified directly from bacterial cultures. Bacterial clones were inoculated in 96-well, 2 ml Assay Block (Corning), containing 600 µl LB/Ampicillin (50 µl /ml) and incubated at 37 °C for 16 hrs. Approximately 1µl of culture suspension was transferred in 96 well plate (Corning), loaded in each single well with 100 µl of the following solution: 1X PCR buffer, 1.5 mM MgCl2, 0.15 mM for each of the four dNTPs, 0.2 µM for each of primer A and B, 0.02U/µl of Taq Polimerase (New England Biolabs). PCR buffer and unincorporated nucleotides were removed by filtering through 96-well multiscreen filter plates (Millipore, Bedford, MA, USA). Spotting was performed using the robotic system Genpak Array 21 (Genetix, Hampshire, UK) equipped with 32 Stealth Micro Spotting Pins SMP 3B (ArrayIt) settled to obtain spots with an average diameter of 120 µm. Samples were spotted in quadruplicate on derivatized glass slides (MICROMAX Glass Slides: SuperChip™ I, PerkinElmer, Wellesley, MA, USA) to obtain more consistent fluorescence measures. Spotting was performed at 50% relative humidity to obtain the best spot morphology and to reduce the plate evaporation. Microarrays were then processed in a UV cross linker (Stratagene, La Jolla, CA, USA) (total power of 300 mJoules) for the binding the DNA to the slides. Microarrays were finally processed to remove any unbound PCR product, dried and stored under vacuum at room temperature in sealed boxes.
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