Printing and Quality Control of the Chip

cDNAs representing ~200 genes were obtained from the NIA Mouse 15K Clone Set and Open Biosystems (Huntsville, Ala). Nucleotides (200-1200 bp) from highly homologous gene-specific regions of mouse and human genes were amplified by PCR using gene specific primers. The quality of PCR product was monitored on agarose gel . Sequence verified PCR products were used for printing. Positive and negative controls were spotted to normalize the sensitivity, signal linearity and consistency of the assay. For example, control spots consist of sequences without homology to any known sequences, thus serving as controls when the corresponding RNA is spiked into the labeling reaction (positive control) or not (negative control).PCR products were printed on UltraGap Gamma Amino Propyl Silane slides (GAPS; Corning Inc). The GAPS surface provides free amino moieties that form ionic attachments to the negatively charged phosphate groups of the DNA backbone. Microarrays were printed using a BioRad VersaArray arrayer using 24 SMP3B stealth pins (TeleChem International Inc). Each cDNA was printed (~125 μm diameter) in duplicate with spot to spot separation of 350 μm.

Quality Control

Several quality control and quality assurance tests are performed per array print run. These tests consist of a nonspecific staining of the printed cDNAs, hybridization with random Cy3 labeled 9mers (Spotcheck, GENPAK Ltd) and experimental hybridizations. This test indicates missing spots and bad morphology. Because it is a hybridization, it also indicates spots that may have poor hybridization. Nonspecific staining is also performed with SYTO-61 Molecular Probes . This stain binds to any nucleic acid bound to the surface and fluoresces under the Cy5 channel of the scanner to identify any features that did not print or which have bad spot morphology. These two approaches, combined with a series of cDNA hybridizations, assess the overall signal of the array as a function of background in both channels.