To test whether our method of integrating data from multiple scans accurately maintained the original microarray intensity information, we conducted a spiked oligo experiment. Oligos designed to hybridize to spots with low expression levels in a specific cDNA sample were labeled and spiked into a hybridization reaction at known concentrations. Cy3-labeled oligos were added as an equimolar mixture, while Cy5-labeled oligos were added as a 5-fold dilution series. In order to perform calculations in the context of actual microarray expression data, oligos corresponding to low intensity spots in the cDNA sample were added to a hybridization reaction containing Cy3- and Cy5-labeled cDNA probes synthesized from the same total yeast RNA sample, a self versus self hybridization. Slides were scanned at a constant laser power (90%) and multiple PMT settings (45-85%), and the data was processed using masliner. Results from this experiment contain two types of data for which the actual ratio is known, ratios of the spiked oligos (present in a five-fold dilution series) and ratios of the self versus self hybridization of the cDNA sample (present at a ratio of one).

Our ability to accurately reconstruct the ratios from the five-fold dilution series of spiked oligos, demonstrates that extrapolation of signal intensities from multiple scans using masliner is accurate over several orders of magnitude.

To further characterize the quality of masliner adjusted values with those acquired from a single scan, we analyzed the values obtained for the remaining 6,298 spots on the spiked oligo arrays which correspond to the cDNA self versus self hybridization. In theory, the gene expression ratio for each of these spots should be one. To visualize the accuracy of the self versus self ratios across the full range of spot intensities from single intensity scans and masliner adjusted values, we used RI plots.

This graph represents a summary of the information contained in an RI plot of self versus self hybridization data. It plots the log of the Cy5:Cy3 ratio versus the sum of the logs of the intensities. Because the a self:self ratio is expected to be 1.0, the spread of along the Y-axis measures the accuracy of the ratio. The spread across the X-axis measures the range of spot intensity detected.

Thn RI plot based on masliner-adjusted scan series of four Cy5 and Cy3 scans (PMTs 45-85). Dots in the RI plot corresponding to array features are represented by different colored symbols depending on which Cy5 and Cy3 PMT scans were used by masliner to pick up for the features; since these depend on spot brightness in the two channels, the RI plot is broken up into different regions.

See a comparison of RI plots of masliner adjusted data and data taken from scans at a single intensity.