> Date: Sun, 20 Nov 2005 10:30:43 +0200 > From: "Ron Ophir" <ron.ophir at="" weizmann.ac.il=""> > Subject: [BioC] [Limma] Correcting Dye swap effect > To: "<bioc mailing="" list"="" <bioconductor="" at="" stat.math.ethz.ch=""> > > Hi, > In Chapter 8.1.2 in Limma users guide there is a description how to > detecting the Dye effect. > The example there describes an experiment of Wt vs Mut with two dye > swaps replicates as follow: > > FileName Cy3 Cy5 > File1 wt mu > File2 mu wt > File3 wt mu > File4 mu wt > > Let's say that I found a list of gene which are significant due to > mutant effect and due to dye effect as well. Can I only ignore them or > can I correct the dye effect? > I guess that it depends how the dye swaps replicates was prepared. If > the dye replicates are technical replicates using the block design is > enough to correct dye effect. If dye replicates are also biological > replicates they night also represent a batch effect. That is all first > replicates was sent in one day and the second replicates sent in another > day, which this difference by itself without dye swap may be a source > for variation. The second option of dye swap preparation may be > corrected by ANCOVA (?). Is possible to do it with LIMMA? I know that a > question about using ANCOVA with LIMMA arose by Naomi Altman but this > discussion was beyond my knowledge. So back to my questions: Is > correcting the dye effect is possible in LIMMA? Yes it is possible to correct the Mu vs WT comparison for the dye- effect, and the analysis given in the Limma User's Guide already does this. If a gene if significant for the Mu vs Wt comparison, this is meaningful even if the same gene also shows a dye- effect. See the limma chapter in the Bioconductor book for a data example. Best wishes Gordon > Is ANCOVA is a solution? > and HOW? > > I hope these questions are in the focus of that mailing list, > Thanks > Ron