Dear all, I analyse affymetrix data to compare transcripts between the wild-type and 3 mutants. After normalization using the RMA method, the histogram of distribution of exprs(eset) isn t normal (cf histesetDataRMA.ps). It seems there two distributions in each array hist. If I continue the analysis using lm.series, all genes are significant. I guess that it remains a variation source which is not normalized. Do you have any idea of what it could be and how I can remove this variation ? The procedure I did: > Data<-ReadAffy() > eset<-rma(Data) > fit<-lm.series(exprs(eset), d1) > eb<-ebayes(fit) > d1 WT A B C Exp143hyb246 1 0 0 0 Exp143hyb247 1 1 0 0 Exp143hyb248 1 0 1 0 Exp143hyb249 1 0 0 1 Exp143hyb410 1 0 0 0 Exp143hyb411 1 0 0 0 Exp143hyb412 1 1 0 0 Exp143hyb413 1 1 0 0 Exp143hyb414 1 0 1 0 Exp143hyb415 1 0 1 0 Exp143hyb416 1 0 0 1 Exp143hyb417 1 0 0 1 > toptable(coeff=2, number=22810, genelist=gnames, fit=fit,eb=eb,adjust="fdr") I tried also the MAS5 procedure to normalize but the histograms of exprs(eset) is more far from the gaussian and all genes are also signficant with the same linear model. Thanks for your answers. Delphine -- ================================================================== Delphine Fleury DEPARTMENT OF PLANT SYSTEMS BIOLOGY Fax:32 (0)9 3313809 GHENT UNIVERSITY/VIB, Technology Park 927, B-9052 Gent, Belgium Vlaams Interuniversitair Instituut voor Biotechnologie VIB mailto:defle@psb.ugent.be http://www.psb.ugent.be ================================================================== -------------- next part -------------- A non-text attachment was scrubbed... Name: histesetDataRMA.ps Type: application/postscript Size: 24747 bytes Desc: not available Url : https://www.stat.math.ethz.ch/pipermail/bioconductor/attachments /20030826/fcd0345f/histesetDataRMA-0001.ps

i dont expect the distribution of exprs(eset) to be normal. why should RNA expression log-scale levels in any tissue follow a normal distribution? some physical models (and SAGE data) suggest expression should follow a power law (very different from a normal distribution) and the results of exprs(eset) are sometimes in agreement with this. what has a chance of being normal is the distribution of expression for the same gene across technical or biological reps. im not sure if the procedure you follow requires these assumptions to hold, but if they do you should look at significance assessments with caution as they are sometimes driven by these assumptions (especially with small amounts of data). permutation tests are an alternative when one cant make these assumptions althought they too can be misleading with small amounts of data. i find looking at volcano plots helpful in these situations. hope this helps, rafael On Tue, 26 Aug 2003, Delphine Fleury wrote: > Dear all, > I analyse affymetrix data to compare transcripts between the wild- type > and 3 mutants. After normalization using the RMA method, the histogram > of distribution of exprs(eset) isn t normal (cf histesetDataRMA.ps). It > seems there two distributions in each array hist. If I continue the > analysis using lm.series, all genes are significant. I guess that it > remains a variation source which is not normalized. Do you have any idea > of what it could be and how I can remove this variation ? > > The procedure I did: > > Data<-ReadAffy() > > eset<-rma(Data) > > fit<-lm.series(exprs(eset), d1) > > eb<-ebayes(fit) > > d1 > WT A B C > Exp143hyb246 1 0 0 0 > Exp143hyb247 1 1 0 0 > Exp143hyb248 1 0 1 0 > Exp143hyb249 1 0 0 1 > Exp143hyb410 1 0 0 0 > Exp143hyb411 1 0 0 0 > Exp143hyb412 1 1 0 0 > Exp143hyb413 1 1 0 0 > Exp143hyb414 1 0 1 0 > Exp143hyb415 1 0 1 0 > Exp143hyb416 1 0 0 1 > Exp143hyb417 1 0 0 1 > > toptable(coeff=2, number=22810, genelist=gnames, > fit=fit,eb=eb,adjust="fdr") > > I tried also the MAS5 procedure to normalize but the histograms of > exprs(eset) is more far from the gaussian and all genes are also > signficant with the same linear model. > > Thanks for your answers. > Delphine > > -- > ================================================================== > Delphine Fleury > DEPARTMENT OF PLANT SYSTEMS BIOLOGY Fax:32 (0)9 3313809 > GHENT UNIVERSITY/VIB, Technology Park 927, B-9052 Gent, Belgium > Vlaams Interuniversitair Instituut voor Biotechnologie VIB > mailto:defle@psb.ugent.be http://www.psb.ugent.be > ================================================================== > >