I am using the Limma package 1.6.1 to normalize Imagene files. Reading the source code I was wondering if there is a particular reason to take the 'Signal Mean' signal values and the 'Background Median' background values from the Imagene files. Why not for instance use the 'Signal Median' column for the signal values? I hope someone can shed a light on these choices so I can explain them to some of my colleagues regards Victor de Jager

Hi Victor, The signal mean is used in order to average the signal across the entire printed spot, thereby taking into account the entire population of hybridizations for that particular feature on the microarray. In an ideal world there would be perfect uniformity in the amount of DNA printed across the entire feature area, but sometimes this is not the case, and can result from variation introduced at any step from the printing through the slide fixation and pre-hybridization. These variations might manifest as "donuts" or halos, comets (not extreme -- most software that checks for feature morphology will throw these out before you get to the normalization step) or more commonly in my experience, localized "clumping" within the feature. Because the assumption is that the feature exclusively represents a unique and specific target for your probe, we sum the intensity of each pixel within the feature and divide by the number of pixels, thereby arriving at the signal mean for the feature, which should be more representative of the total hybridization events than the median. The background is treated differently because the assumption here is that there should be no DNA present in the areas between the features, and therefore there should be no specific hybridization taking place. However, again because of random variation that can be introduced throughout the handling of the slide over the entire course of the experiment, there may be isolated pockets of increased non-specific background at various points on the slide. Generally, because the number of pixels in a given spot's background having an elevated non-specific signal is relatively few compared to the total number of background pixels, if one were to take the background mean, these spurious non-specific signals might contribute to a (falsely) higher background level than if one would simply take the median intensity for all the pixels represented in the background and should therefore be more representative of the actual background signal. Regards, John. -----Original Message----- From: bioconductor-bounces@stat.math.ethz.ch [mailto:bioconductor-bounces@stat.math.ethz.ch] On Behalf Of V.C.L. de Jager Sent: Tuesday, May 11, 2004 9:30 AM To: bioconductor@stat.math.ethz.ch Subject: [BioC] Limma package question I am using the Limma package 1.6.1 to normalize Imagene files. Reading the source code I was wondering if there is a particular reason to take the 'Signal Mean' signal values and the 'Background Median' background values from the Imagene files. Why not for instance use the 'Signal Median' column for the signal values? I hope someone can shed a light on these choices so I can explain them to some of my colleagues regards Victor de Jager _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor