Microarray Scanner Calibration Curves: Characteristics and Implications
Author Information
Author(s): Shi Leming, Tong Weida, Su Zhenqiang, Han Tao, Han Jing, Puri Raj K, Fang Hong, Frueh Felix W, Goodsaid Federico M, Guo Lei, Branham William S, Chen James J, Xu Z Alex, Harris Stephen C, Hong Huixiao, Xie Qian, Perkins Roger G, Fuscoe James C
Primary Institution: National Center for Toxicological Research, U.S. Food and Drug Administration
Hypothesis
How do different PMT gains affect the calibration curves of Cy5 and Cy3 dyes in microarray scanners?
Conclusion
Scanning microarray slides at fixed, optimal gain settings maximizes the linearity between concentration and intensity, improving accuracy.
Supporting Evidence
- Calibration curves for the same dye under the same PMT gain are nonlinear at both high and low intensity ends.
- The degree of nonlinearity of the calibration curve depends on the PMT gain.
- The background intensity for the Cy3 channel is higher than that for the Cy5 channel.
- Lowess normalization improves reproducibility but does not fully correct systematic deviation from true ratios.
Takeaway
This study shows that using the right settings on a microarray scanner helps get better and more accurate results when measuring gene activity.
Methodology
The study evaluated calibration curves for Cy5 and Cy3 dyes under 18 PMT gains using a calibration slide with known dye concentrations.
Potential Biases
Potential bias due to differences in background fluorescence and non-specific binding.
Limitations
The study primarily focuses on two dyes and may not generalize to all microarray technologies.
Digital Object Identifier (DOI)
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