研究目的
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.
研究成果
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.
研究不足
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.
1:Experimental Design and Method Selection:
The study involved manufacturing DNA microarray slides with immobilized cyanine-labeled single-strand DNA, scanning them multiple times with varying PMT voltages to observe photobleaching effects, and developing a mathematical model based on degradation processes.
2:Sample Selection and Data Sources:
Single-strand DNAs of 40 nt length with Cy3 or Cy5 modifications were purchased and serially diluted. Microarray slides were prepared with identical layouts.
3:List of Experimental Equipment and Materials:
Equipment includes a GenePix 4000B Microarray Scanner, Nano Plotter NP
4:1 non-contact spotter, aldehyde glass slides (SuperAldehyde 2), and software like GenePix Pro 0, R Studio, and MATLAB. Materials include cyanine dyes, buffers (e.g., SSC, CHAPS), and DNA oligos. Experimental Procedures and Operational Workflow:
Oligos were spotted onto slides, dried, washed, and scanned repeatedly with constant or varying PMT voltages at 100% laser power. Data was collected and filtered for quality.
5:Data Analysis Methods:
Data was analyzed using non-linear least-square fitting in MATLAB, ANOVA for parameter dependencies, and linear fits for validation. Background correction was omitted to avoid additional variability.
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