Hi Emanuele, marchiem@libero.it wrote: > Hi Wolfgang, > first of all thank you very much for your quick reply... > we use gDNA as reference because our lab want to compare our data with other labs which use the same design. We have data express in in gDNA units... > We normalize these data with Genespring using a globlal normalization method that calculate the scaling factor from the 50th percentile of genes. So then why not use a global scaling to the median with bioconductor as well? There are options for that in the normalization procedures of the 'marray' and 'limma' packages. > ...but if I have a time course i don't think I can use these single channels methods to normalize, am I wrong? If I don't consider the reference channel I could confound a brighter array as a more expressed...how can I distinguish that? > sorry if I am too trivial and thanks again! I am not sure I understand. It is the purpose of normalization methods to correct for some arrays being more or less bright, and the ones that I mentioned do so. They rely on some assuptions: - e.g. median normalization assumes that the median intensity for each array should be the same - e.g. vsn requires that there is a set of >50% of genes on the array that are approximately not differentially expressed (in the non gDNA channel) - e.g. quantile normalization assumes that the true distribution of intensities is the same on all arrays Whether that holds for your data you need to check. I don't think it has anything to do with whether you're looking at a time series or something else. Best wishes Wolfgang ------------------------------------- Wolfgang Huber Division of Molecular Genome Analysis German Cancer Research Center Heidelberg, Germany Phone: +49 6221 424709 Fax: +49 6221 42524709 Http: www.dkfz.de/abt0840/whuber