Scanner calibration revisited

BACKGROUND: Calibration of a microarray scanner is critical for accurate interpretation of microarray results. Shi et al. (BMC Bioinformatics, 2005, 6, Art. No. S11 Suppl. 2.) reported usage of a Full Moon BioSystems slide for calibration. Inspired by the Shi et al. work, we have calibrated microarray scanners in our previous research. We were puzzled however, that most of the signal intensities from a biological sample fell below the sensitivity threshold level determined by the calibration slide. This conundrum led us to re-investigate the quality of calibration provided by the Full Moon BioSystems slide as well as the accuracy of the analysis performed by Shi et al. METHODS: Signal intensities were recorded on three different microarray scanners at various photomultiplier gain levels using the same calibration slide from Full Moon BioSystems. Data analysis was conducted on raw signal intensities without normalization or transformation of any kind. Weighted least-squares method was used to fit the data. RESULTS: We found that initial analysis performed by Shi et al. did not take into account autofluorescence of the Full Moon BioSystems slide, which led to a grossly distorted microarray scanner response. Our analysis revealed that a power-law function, which is explicitly accounting for the slide autofluorescence, perfectly described a relationship between signal intensities and fluorophore quantities. CONCLUSIONS: Microarray scanners respond in a much less distorted fashion than was reported by Shi et al. Full Moon BioSystems calibration slides are inadequate for performing calibration. We recommend against using these slides.