[BioC] M vs M plots- dye effect in dyeswap exp

[Sean Davis]

On 2/6/06 11:19 AM, "lzarzabal at aol.com" <lzarzabal at aol.com> wrote:

> Hello All,
>  
> I was wondering if anyone had any suggestions on quality issues with dyeswap
> slides.  We ran 2 exp's with dye swap slides and some of them looked
> questionable.  I have attached the M vs M plots.  Also several of the density
> plots are bimodal.  We had missing spots that were missed by the print tips,
> which I removed. I read in previous BioC emails that if there is an x pattern
> on the MvsM plot that it could be due to degradation of the dyes.  The
> experimenters have assured me that the dyes are not expired and are from a
> very reputable company.  Does anyone have any ideas, are the slides usable or
> need to be redone?  We are using in-house RNA.

I would spend some time looking at the quality of your individual arrays.
Are the density plots all similar?  What do the individual-array scatter
plots look like?  What are the mean/median intensities in each channel when
compared to each other?  How about compared to other arrays?  How about
background across the arrays?  Does hierarchical clustering show any
systematic biases such as date, print batch, RNA extraction, etc.?  Try
answering some of these questions first, and then your answer to why dye
swaps "look questionable" might be more apparent.

Sean