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.
The application of DNA microarrays for high throughput analysis of genetic regulation is often limited by the fluorophores used as markers. The implementation of multi-scan techniques is limited by the fluorophores’ susceptibility to photobleaching when exposed to the scanner laser light. This paper presents combined mechanical and chemical strategies which enhance the photostability of cyanine 3 and cyanine 5 as part of solid state DNA microarrays. These strategies are based on scanning the microarrays while the hybridized DNA is still in an aqueous solution with the presence of a reductive/oxidative system (ROXS). Furthermore, the experimental setup allows for the analysis and eventual normalization of Förster-resonance-energy-transfer (FRET) interaction of cyanine-3/cyanine-5 dye combinations on the microarray. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the comparability of microarray experiment results between labs.
Desoxyribonucleic acid (DNA) microarray experiments generate big datasets. To successfully harness the potential information within, multiple filtering, normalization, and analysis methods need to be applied. An in-depth knowledge of underlying physical, chemical, and statistical processes is crucial to the success of this analysis. However, due to the interdisciplinarity of DNA microarray applications and experimenter backgrounds, the published analyses differ greatly, for example, in methodology. This severely limits the comprehensibility and comparability among studies and research fields. In this work, we present a novel end-user software, developed to automatically filter, normalize, and analyze two-channel microarray experiment data. It enables the user to analyze single chip, dye-swap, and loop experiments with an extended dynamic intensity range using a multiscan approach. Furthermore, to our knowledge, this is the first analysis software solution, that can account for photobleaching, automatically detected by an artificial neural network. The user gets feedback on the effectiveness of each applied normalization regarding bias minimization. Standardized methods for expression analysis are included as well as the possibility to export the results in the Gene Expression Omnibus (GEO) format. This software was designed to simplify the microarray analysis process and help the experimenter to make educated decisions about the analysis process to contribute to reproducibility and comparability.
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