Human Array 2.0 was constructed arraying on glass slides cDNA inserts corresponding to the 3’-portion of mRNAs, from a collection of 4,670 different bacterial clones. This collection was obtained by the systematic sequencing of human skeletal muscle, heart and hematopoietic cell specific libraries. 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). The purification protocol was automated using the 96 channel robotic workstation Multimek (Beckman) to avoid errors due to the manual handling of large number of samples. Quality control and quantification of the amplified products was done by separation of 3 µl samples on agarose gels containing EtBr and gel image analysis with Chemi Doc UV transilluminator equipped with the Quantity One software (Bio-Rad, Hercules, CA, USA). PCR products were then lyophilized and plates were sealed using thermowell sealers (Corning) and stored at –20°C. For microarray printing, PCR products were dissolved in 15 µl of Micro Spotting Solution (ArrayIt, Telechem, Sunnyvale, CA, USA) by vigorous shaking of plates for 4-5 hrs at 4°C, and transferred in 384 well plates. 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 duplicate 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. Keywords = cDNA microarray Keywords = human skeletal muscle Keywords = heart Keywords = bone marrow Keywords = EST
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