[BioC] [Limma] Correcting Dye swap effect

[Gordon K Smyth]

> 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