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Sources of nonlinearity in cDNA microarray expression measurements.

L Ramdas¹, K R Coombes, K Baggerly

¹Department of Pathology, University of Texas M D Anderson Cancer Center, Houston, TX 77030, USA. wzhang@mdanderson.org

|December 12, 2001

Summary

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Microarray data analysis assumes linear signal-to-gene expression. This study found nonlinearities in both radioactive and fluorescent microarrays due to signal quenching and scanner transformation, impacting gene expression analysis.

Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

Microarray data analysis relies on the assumption of a linear relationship between quantified signal intensity and gene expression levels.
This assumption is critical for accurate interpretation of gene expression data derived from microarrays.

Purpose of the Study:

To experimentally validate the linearity assumption in microarray data analysis.
To investigate potential sources of nonlinearity in both radioactive and fluorescent microarray platforms.

Main Methods:

Examined the signal-to-expression relationship for radioactively labeled cDNA on nylon membranes.
Investigated the signal-to-expression relationship for fluorescently labeled cDNA on glass slides.
Utilized dilution series experiments to detect nonlinearities.

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Main Results:

Identified signal quenching due to excessive dye concentrations as a source of nonlinearity in fluorescent microarrays.
Discovered a nonlinear data transformation by the scanner affecting radioactive microarrays.
Noted that image-quantification software packages varied in their correction for scanner-induced nonlinearities.

Conclusions:

Two sources of nonlinearity were identified, impacting both radioactive and fluorescent microarray analyses.
The scanner-induced nonlinearity in radioactive microarrays is particularly problematic as it is not easily predictable.
Simple dilution series are recommended as a quality control measure to detect these nonlinearities.