(sorry, forgot to CC to the list.) Dear Edwin, first of all, the code you have posted cannot work because it never assigns the results of your computations to any variables. In order to help you, we need to know at least 1) what code you have really run, 2) your refdesign matrix, 3) the values from topTable() and RG.MA() for at least one example probe, 4) HOW you backcalculated R, G, and FC from topTable(), and 5) the obligatory output of sessionInfo() although this doesn't really sound like a version issue. :-) AFAIK, eBayes() will affect the t, p, and B statistics computed from your M values but not the AveExpr and logFC. RG.MA() backcalculates background-adjusted and normalized R and G values, so I am not sure what you mean by "observed" RG -- you don't get back the original R, Rb, G, Gb from an MAList via RG.MA(). Cheers, - axel Axel Klenk Research Informatician Actelion Pharmaceuticals Ltd / Gewerbestrasse 16 / CH-4123 Allschwil / Switzerland "Edwin Groot" <edwin.groot at="" biol="" ogie.uni-freiburg="" to="" .de=""> bioconductor at stat.math.ethz.ch Sent by: cc bioconductor-boun ces at stat.math.eth Subject z.ch [BioC] LIMMA: Why does eBayes expression differ from observed? 30.07.2009 12:38 I am getting odd results from a common reference design analysis of two-colour data in LIMMA. Previously I had analyzed only simple-comparison designs. Hopefully you can help restore credibility of Bioconductor to my supervisor. Why does the AveExpr and logFC reported in topTable() differ from the replicates in my MA object? The analysis is a textbook example of comparing a series of mutants to the wild type. Wild type is always green. The summary is as follows: lmfit(MA, refdesign) eBayes(fit) topTable(fit, coef="mut1") #Regenerate the RG from MA RG.MA(MA) Backcalculating the topTable AveExpr and logFC to green, red and FC gives the following expressions as an example: WT: 31 mut1: 298 FC: 9.5 Compare that to the average of 9 WT and 3 mut1 in the RG.MA(MA) list: WT: 58 mut1: 611 FC: 10.5 A survey of other probes gives an over and underestimate of the observed RG from 1.5 to 5 times. What is the explanation for that? What should I troubleshoot, besides looking at the usual BG and FG distributions and MA plots? Regards, Edwin --- Dr. Edwin Groot, postdoctoral associate AG Laux Institut fuer Biologie III Schaenzlestr. 1 79104 Freiburg, Deutschland +49 761-2032945 _______________________________________________ Bioconductor mailing list Bioconductor at stat.math.ethz.ch 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