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Microarray scanner calibration curves: characteristics and implications.
Leming Shi1, Weida Tong, Zhenqiang Su
1National Center for Toxicological Research, U.S. Food and Drug Administration, 3900 NCTR Road, Jefferson, Arkansas 72079, USA. leming.shi@fda.hhs.gov
BMC Bioinformatics
|July 20, 2005
Summary
Microarray calibration curves are nonlinear, impacting mRNA abundance accuracy. Optimal scanner gain settings and a new ratio calculation method are proposed to improve data reliability.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Microarray analysis assumes linear dye concentration to fluorescence intensity.
- Real-world calibration curves often exhibit nonlinearity.
Purpose of the Study:
- Evaluate nonlinear calibration characteristics of Cy5 and Cy3 dyes.
- Assess the impact of nonlinearity on mRNA abundance measurements.
- Propose methods to correct ratio bias in microarray data.
Main Methods:
- Scanning microarray calibration slides at 18 PMT gains.
- Analyzing calibration curves for Cy5 and Cy3.
- Utilizing simulation results.
- Demonstrating Lowess normalization effects.
- Proposing a new ratio calculation method.
Main Results:
- Calibration curves are nonlinear at high and low intensities.
- Nonlinearity varies with PMT gain and differs between Cy5 and Cy3.
- Cy3 channel has higher background intensity.
- Nonlinearity causes ratio underestimation and intensity-dependent bias.
- Lowess normalization improves reproducibility but not accuracy.
Conclusions:
- Scan microarrays at fixed, optimal PMT gains for maximal linearity.
- Normalization improves reproducibility but not accuracy.
- A new ratio calculation method is proposed for bias correction.