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The Impact of Photobleaching on Microarray Analysis

DOI:10.3390/biology4030556 期刊:Biology 出版年份:2015 更新时间:2025-09-23 15:22:29
摘要: 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.
作者: Marcel von der Haar,John-Alexander Preu?,Kathrin von der Haar,Patrick Lindner,Thomas Scheper,Frank Stahl
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To characterize and quantify the impact of photobleaching on DNA microarray analysis, specifically for cyanine-3 and cyanine-5 fluorophores, and to develop a model to correct for photobleaching-induced bias in multi-scan procedures.

The developed models for Cy3 and Cy5 photobleaching effectively predict signal intensity reduction based on initial intensity, number of scans, and PMT voltage, allowing for bias correction in multi-scan procedures. This improves data quality and reproducibility, contributing to standardization in microarray analysis. Future work should validate the models with actual hybridization experiments and address additional factors like background intensity and environmental influences.

The study uses directly labeled ssDNA without hybridization, which does not reflect real microarray conditions involving cDNA hybridization. Effects like FRET, intra-spot heterogeneity, and environmental factors (e.g., ozone, light exposure) were not fully accounted for. Background photobleaching was not investigated, and the model may not generalize to all microarray setups.

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