Sources of nonlinearity in cDNA microarray expression measurements.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Ramdas
L
L
Department of Pathology, University of Texas M D Anderson Cancer Center, Houston, TX 77030, USA. wzhang@mdanderson.org
Coombes
K R
KR
Baggerly
K
K
Abruzzo
L
L
Highsmith
W E
WE
Krogmann
T
T
Hamilton
S R
SR
Zhang
W
W
D002138:Calibration; D005453:Fluorescence; D020869:Gene Expression Profiling; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011786:Quality Control; D011851:Radioactivity; D012015:Reference Standards; D015398:Signal Transduction; D012984:Software