TY - JOUR
T1 - Sources of nonlinearity in cDNA microarray expression measurements
AU - Ramdas, Latha
AU - Coombes, Kevin R.
AU - Baggerly, Keith
AU - Abruzzo, Lynne
AU - Highsmith, W. Edward
AU - Krogmann, Tammy
AU - Hamilton, Stanley R.
AU - Zhang, Wei
N1 - Publisher Copyright:
© 2001, Ramdas et al., licensee BioMed Central Ltd.
PY - 2001/11
Y1 - 2001/11
N2 - 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.
AB - 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.
KW - Dilution Experiment
KW - Microarray Image
KW - Serial Dilution Experiment
KW - Storm PhosphorImager
KW - cDNA Microarray Expression
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U2 - 10.1186/gb-2001-2-11-research0047
DO - 10.1186/gb-2001-2-11-research0047
M3 - Article
C2 - 11737946
AN - SCOPUS:0035233680
SN - 1474-7596
VL - 2
JO - Genome Biology
JF - Genome Biology
IS - 11
M1 - research0047.1
ER -