Roger Vallejo wrote: > This must be of interest for those preprocessing data from > affymetrix chips. We have compared RMA vs VSN performing an > lme-ANOVA. If you are wondering what to use RMA or VSN? or what > are the potential pitfalls or benefits from using either > normalization or background data correction > approach. Then, please read below and make your own conclusions. Hi Roger, I don't get your point. As Rafael has already pointed out, vsn is a normalization method, while RMA is a combination of PM/MM synthesis, normalization, and probe set summary. In the vsn vignette I give an example how vsn can be used instead of quantile normalization as a normalization method within RMA (through the espresso function). The example is simple-minded, since it ignores the MMs! This is better than subtracting (PM-MM), but there are smarter ways to use the MMs, e.g. in GCRMA. When I made the example, it was interesting to see that a parametric normalization method like vsn could in many cases outperform a non-parametric method method like quantile normalization. But affy preprocessing is a combination of several steps, and especially the background correction (PM/MM synthesis) seems to benefit a lot from fine-tuning - see Rafa's and coworkers' recent work. Also, when you try to estimate fold-changes from weakly expressed genes, you will always have to deal with the variance-bias trade-off. The relative measurement error in weak probe signals is large, and thus log-ratios between them can fluctuate strongly. Different algorithms make different choices about how to deal with them. What is best for you depends on the question of interest and the experimental design. E.g. when you have a very large number of replicates you don't want shrinkage, while with a small number of replicates, shrinkage is often beneficial. 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