Measuring absolute expression with microarrays with a calibrated reference sample and an extended signal intensity range

Gene expression ratios derived from spotted-glass microarray experiments have become invaluable to researchers by providing sensitive and comprehensive indicators of the molecular underpinnings of cell behaviors and states. However, several drawbacks to this form of data have been noted, including the inability to relate ratios to absolute expression levels or to compare experimental conditions not measured with the same control. In this study we demonstrate a method for overcoming these obstacles. First, instead of cohybridizing labeled experimental and control samples, we hybridize each sample against labeled oligos complementary to every microarray feature. Ratios between sample intensities and intensities of the oligo reference measure sample RNA levels on a scale that relates to their absolute abundance, instead of to the variable and unknown abundances of a cDNA reference. We demonstrate that results from this type of hybridization are accurate and retain absolute abundance information far better than conventional microarray ratios. Next, to ensure the accurate measurement of sample and oligo reference intensities, which may differ by several orders of magnitude, we use a linear regression algorithm, implemented in a freely available PERIL script, to combine the linear ranges of multiple scans taken at different scanner sensitivity settings onto an extended linear scale. We discuss future applications of our method to measure RNA expression on the absolute scale of number of transcripts per cell from any organism for which oligo-based spotted-glass microarrays are available.