The 7.7k human cDNA arrays used here are part of a larger collection of 30k cDNAs purchased from Research Genetics (Huntsville, AL). Plasmids were isolated from overnight bacterial cultures in 96-well plates and cDNA inserts amplified by PCR using M13 primers 5'-gtt gta aaa cga cgg cca gtg and 5'-cac aca gga aac agc tat g. The PCR products were precipitated with ethanol, washed and redissolved in 3x SSC (sodium dodecyl sulphate). Quality and concentration was assessed by agarose gel electrophoresis. The purified PCR products were prepared in 50% DMSO (about 50-200 ng/ l) and spotted in duplicate onto 25 x 75mm amino-silane coated CMT-GAPS glass slides (Corning, NY) using a MicroGrid II printing robot (BioRobotics; Cambridge, UK). The arrays were spotted with 8 pins and contained 4 x 8 blocks of 22 x 22 spots. The probes were fixed to the slide surface by UV irradiation. The 7.7k cDNA probes represent 7561 unique UniGene clusters, all printed in duplicates. In addition, a few in-house cDNA probes and a set of positive and negative control elements including 10 different PCR products of exogenous DNA from SpotReport™ cDNA Array Validation System (Stratagene, La Jolla, CA) were printed on the arrays. Totally, each array contained 15488 elements including duplicates and controls.
