The Impact of Photobleaching on Microarray Analysis

Abstract

DNA-Microarrays have become a potent technology for high-throughput analysis of genetic regulation. However, the wide dynamic range of signal intensities of fluorophore-based microarrays exceeds the dynamic range of a single array scan by far, thus limiting the key benefit of microarray technology: parallelization. The implementation of multi-scan techniques represents a promising approach to overcome these limitations. These techniques are, in turn, limited by the fluorophores’ susceptibility to photobleaching when exposed to the scanner’s laser light. In this paper the photobleaching characteristics of cyanine-3 and cyanine-5 as part of solid state DNA microarrays are studied. The effects of initial fluorophore intensity as well as laser scanner dependent variables such as the photomultiplier tube’s voltage on bleaching and imaging are investigated. The resulting data is used to develop a model capable of simulating the expected degree of signal intensity reduction caused by photobleaching for each fluorophore individually, allowing for the removal of photobleaching-induced, systematic bias in multi-scan procedures. Single-scan applications also benefit as they rely on pre-scans to determine the optimal scanner settings. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the lab-to-lab comparability of microarray experiment results.

Optimization of Cyanine Dye Stability and Analysis of FRET Interaction on DNA Microarrays

“…These findings are confirmed in this study, as photobleaching and FRET result in significantly different data: as shown in Figure 4(1a,b), without protective measures, photobleaching occurs similar to previous findings of von der Haar et al [19] with intensity decreases for Cy3 and to a higher degree for Cy5. Interestingly, the 96 gene experiment shows that these photobleaching percentages nearly switch when applying a wet chamber with ROXS.…”

“…Next to biological and biochemical sources, bias originates from photochemical processes and depends on the choice of labeling agent as well as the selected imaging procedure and environment. In earlier works, it was shown that the ubiquitous application of cyanine dye labeling causes significant bias due to the dyes’ disparate susceptibility to photobleaching and possible FRET interaction [19,20,26,28,29]. …”

“…Based on the findings of Vogelsang et al [20], Rao et al [26], and our own previous research [19], a novel strategy for the minimization of photobleaching in cyanine-labeling-based DNA microarray experiments was successfully implemented. The modification of DNA microarray slides with thin liquid chambers filled with a buffer containing ROXS provided a valid protection of cyanine dyes against photo destruction occurring in the scanning process.…”

“…Its effect on scanner data was successfully characterized and an empirical model was devised. The model efficiently normalizes the bias introduced by this effect with respect to the choice of dye, the previously carried out scans, and the scanner settings [19]. This study aims to minimize photobleaching of Cy3 and Cy5 using a reductive-oxidative, protective buffer (ROXS).…”

Array Analysis Manager—An automated DNA microarray analysis tool simplifying microarray data filtering, bias recognition, normalization, and expression analysis

“…Cyanine‐3 (Cy3) and cyanine‐5 (Cy5) are ubiquitously used for nucleotide labeling. In previous works, we showed that they possess a varying degree of susceptibility toward photobleaching . All these findings imply an effect of photobleaching that will be even more significant on multiscan data quality.…”