Signal stability of Cy3 and Cy5 on antibody microarrays

Abstract

Background: The antibody microarray technique is a newly emerging proteomics tool for differential protein expression analyses that uses fluorescent dyes Cy 3 and Cy 5. Environmental factors, such as light exposure, can affect the signal intensity of fluorescent dyes on microarray slides thus, it is logical to scan microarray slides immediately after the final wash and drying processes. However, no research data are available concerning time-dependent changes of fluorescent signals on antibody microarray slides to this date. In the present study, microarray slides were preserved at -20 degrees C after regular microarray experiments and were rescanned at day 10, 20 and 30 to evaluate change in signal intensity.

Results: Fluorescent intensities of microarray spots were detected using a confocal laser scanner after the experiment at day 0, and re-examined at day 10, 20 and 30, respectively. Fluorescent intensities of rescanned microarray spots did not show significant changes when compared with those scanned immediately after standard microarray experiments.

Conclusion: Microarray slides can be preserved and rescanned multiple times using a confocal laser scanner over a period of days or weeks.

Figure 1

Examples of Cy3 signals on a microarray slide scanned atday 0, 10, 20, and 30, respectively. At day 0, the spot intensities on this slide range from 261 to 46409, with an average background intensity of 366 versus an average intensity of 488 for the 4 negative control spots. The scale bar on the right indicates color-coded signal intensities.

Figure 2

Examples of Cy5 signals on a microarray slide scanned at day 0, 10, 20, and 30, respectively. At day 0, the spot intensities on this slide range from 592 to 58229, with an average background intensity of 2504 versus an average intensity of 2512 for the 4 negative control spots. The scale bar on the right indicates color-coded signal intensities.