Dear BioC, I have a pretty straight two-color microarray question. My data is in .gpr format, which I have attempted to normalize using the limma and limmaGUI packages in R. My output for each array consists of one value per gene scaled to zero for BOTH CY3 and CY5 combined. Is this expected? I had anticipated my output would consist of separate values for each dye and that they would be log2 fold intensities (similar to single color arrays). I have not been able to find much info regarding this. Any insight will be greatly appreciated. Thanks, Bryan [[alternative HTML version deleted]]

Dear Bryan, when working with two-color arrays, you're typically using the log2 fold change M ( log2(CY5) - log2(CY3) ) and the average intensity A ( (log2(CY5) + log2(CY3))/2 ) instead of separate intensities by channel and limma's normalization functions will do the conversion for you, see ?normalizeWithinArrays and/or Ch. 6 of limmaUsersGuide(). If you want to have normalized intensities, you can back-transform the normalization results using ?RG.MA (note that the resulting values will be unlogged). Limma can also perform separate channel analysis of two-color data, see Ch. 9 of the user's guide if that's what you want. Cheers, - axel Axel Klenk Research Informatician Actelion Pharmaceuticals Ltd / Gewerbestrasse 16 / CH-4123 Allschwil / Switzerland From: Bryan Cassone <bcassone at="" nd.edu=""> To: <bioconductor at="" stat.math.ethz.ch=""> Date: 20.07.2011 21:42 Subject: [BioC] two color microarray normalization Sent by: bioconductor-bounces at r-project.org Dear BioC, I have a pretty straight two-color microarray question. My data is in .gpr format, which I have attempted to normalize using the limma and limmaGUI packages in R. My output for each array consists of one value per gene scaled to zero for BOTH CY3 and CY5 combined. Is this expected? I had anticipated my output would consist of separate values for each dye and that they would be log2 fold intensities (similar to single color arrays). I have not been able to find much info regarding this. Any insight will be greatly appreciated. Thanks, Bryan [[alternative HTML version deleted]] _______________________________________________ Bioconductor mailing list Bioconductor at r-project.org https://stat.ethz.ch/mailman/listinfo/bioconductor Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor The information of this email and in any file transmitted with it is strictly confidential and may be legally privileged. It is intended solely for the addressee. If you are not the intended recipient, any copying, distribution or any other use of this email is prohibited and may be unlawful. In such case, you should please notify the sender immediately and destroy this email. The content of this email is not legally binding unless confirmed by letter. Any views expressed in this message are those of the individual sender, except where the message states otherwise and the sender is authorised to state them to be the views of the sender's company. For further information about Actelion please see our website at http://www.actelion.com