Abstract
BackgroundCommercial microarray scanners and software cannot distinguish between spectrally overlapping emission sources, and hence cannot accurately identify or correct for emissions not originating from the labeled cDNA. We employed our hyperspectral microarray scanner coupled with multivariate data analysis algorithms that independently identify and quantitate emissions from all sources to investigate three artifacts that reduce the accuracy and reliability of microarray data: skew toward the green channel, dye separation, and variable background emissions.ResultsHere we demonstrate that several common microarray artifacts resulted from the presence of emission sources other than the labeled cDNA that can dramatically alter the accuracy and reliability of the array data. The microarrays utilized in this study were representative of a wide cross-section of the microarrays currently employed in genomic research. These findings reinforce the need for careful attention to detail to recognize and subsequently eliminate or quantify the presence of extraneous emissions in microarray images.ConclusionHyperspectral scanning together with multivariate analysis offers a unique and detailed understanding of the sources of microarray emissions after hybridization. This opportunity to simultaneously identify and quantitate contaminant and background emissions in microarrays markedly improves the reliability and accuracy of the data and permits a level of quality control of microarray emissions previously unachievable. Using these tools, we can not only quantify the extent and contribution of extraneous emission sources to the signal, but also determine the consequences of failing to account for them and gain the insight necessary to adjust preparation protocols to prevent such problems from occurring.
Highlights
Commercial microarray scanners and software cannot distinguish between spectrally overlapping emission sources, and cannot accurately identify or correct for emissions not originating from the labeled cDNA
We have developed a hyperspectral-imaging microarray scanner [18] that allows the simultaneous quantification of all fluorescent species, including the spot-localized background leading to a significant improvement in the accuracy of microarray data
The CCD readout rate is the limiting factor in the current hyperspectral scanner (HSS) and newly available charge-coupled device (CCD) detector electronics could allow the HSS to scan at speeds up to twice the speed of commercial scanners
Summary
Commercial microarray scanners and software cannot distinguish between spectrally overlapping emission sources, and cannot accurately identify or correct for emissions not originating from the labeled cDNA. Non-biological factors including printing artifacts, dye-gene interactions, background emissions, and slide-to-slide variations significantly reduce the ability to accurately monitor changes in gene expression in microarray experiments [4,7,8,9,10] These experimental factors are common and often laboratory dependant due to the complicated multi-step procedures used in the production, hybridization, and analysis of microarrays. Various background correction methods exist in data analysis software, but all of these methods make one critical assumption about the data – that the background emissions are the same outside the spot as they are under the spot This assumption is valid in an ideal situation where a perfectly homogeneous glass slide is the sole source of background emissions and the printed DNA spot is sufficiently thin and non-scattering so as not to interfere with the excitation of the glass beneath it. Researchers have shown the results of microarray experiments are very dependant on background subtraction methods used and have theorized that local background values are not representative of the true
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