Sources of nonlinearity in cDNA microarray expression measurements
1 Department of Pathology, University of Texas M D Anderson Cancer Center, Houston, TX 77030, USA
2 Department of Biostatistics, University of Texas M D Anderson Cancer Center, Houston, TX 77030, USA
3 Department of Biomathematics, University of Texas M D Anderson Cancer Center, Houston, TX 77030, USA
4 Department of Hematopathology, Cancer Genomics Core Laboratory, University of Texas M D Anderson Cancer Center, Houston, TX 77030, USA
5 Department of Pathology, University of Maryland, College Park, MD 20742, USA
Genome Biology 2001, 2:research0047-research0047.7 doi:10.1186/gb-2001-2-11-research0047Published: 18 October 2001
A key assumption in the analysis of microarray data is that the quantified signal intensities are linearly related to the expression levels of the corresponding genes. To test this assumption, we experimentally examined the relationship between signal and expression for the two types of microarrays we most commonly encounter: radioactively labeled cDNAs on nylon membranes and fluorescently labeled cDNAs on glass slides.
We uncovered two sources of nonlinearity. The first, which led to discrepancies in analysis affecting the fluorescent signals, was signal quenching associated with excessive dye concentrations. The second, affecting the radioactive signals, was a nonlinear transformation of the raw data introduced by the scanner. Correction for this transformation was made by some, but not all, image-quantification software packages.
The second type of nonlinearity is more troublesome, because it could not have been predicted a priori. Both types of nonlinearities were detected by simple dilution series, which we recommend as a quality-control step.