Abstract
Abstract cDNA microarray contains densely packed spots with known DNA sequences. The deposited DNA sample is referred to as the probe. After the fluorescently-labeled target DNA is hybridized to its complementary probe strand, the microarray was scanned to measure the intensity of the fluorescent signals on each spot. The intensity of each spot represents the expression level of a specific gene. However, the DNA distribution within each spot will influence the analysis of the fluorescent signals. This research aims at applying the spectral images technology and other analytical methods to study the distribution of DNA within the probe spot and analyze the cause of its irregular distribution. The spectral-scanning system was applied in this study to scan the spectrum of probes labeled with Cy3 dye on the cDNA microarray. The characteristics of spectrum were used to distinguish the fluorescence, which may overlap with that of Cy3, emitted from the other materials on the cDNA microarray. SSC, which was added in the DNA spotting solution, do not produce fluorescent signal, therefore cannot be detected by the fluorescence and spectral imaging systems. The non- fluorescence morphologic image acquired by EMCCD camera represents the distribution of crystal, after the spotting solution was dry out. We compared the morphologic and fluorescent images of each spot. The pixel correlation between the distribution of DNA and crystal is higher than 0.9. Furthermore, data reveals that rehydration and surface blocking also affected the distribution of crystal and Cy3-labeled DNA on the glass surface. When microarray slides were processed by rehydration followed by snap-heating, UV cross-linking and baking, the SSC and DNA crystal diffused outward and crystallized out DNA on its surface. DNA molecules tend to gather inward, thus the spots become more rounded, enlarged and the DNA is more evenly distributed. After the surface blocking and washing, DNA molecules and SSC crystal that were loosely attached to the glass surface were washed away. Therefore, the distribution of DNA became less homogenized. The degree of homogenization of DNA distribution on the glass surface affects the reliability of cDNA microarray results.