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

[zarzabal]

Thanks for replying.
I was assured that these pairs are dyeswaps.
Yes, normalization was performed and the scatter plots look good after 
normalization.

As Sean suggested I went back and reviewed all my plots. The density 
plots for the kidney arrays look very similar but the bimodal peak is 
more defined in the kidney arrays. For the liver arrays the plots are 
less similar across arrays and only a couple still have the bimodal peak 
after normalization. The individual scatter plots look pretty good after 
normalization, but the dyeswap slides do not look like mirror images of 
each other-shouldn't they?. Some almost look identical. The background 
images all seem to favor the green dye more than red and there is 
definitely a spatial artifact from the missing spots in the center.

I also reviewed the mean/median intensities of both the raw intensities 
(red and green) and the normalized log ratio values. The values for each 
channel looks pretty comparable across arrays. Of course I am very new 
at this so I am not sure what sizable differences are acceptable or not. 
I am curious though, shouldn't my median values for each dyeswap pair be 
close? There was a very big difference on a few of the pairs.

As for the clustering suggestion, I do not have all the data, (print 
batch, RNA extraction) needed.

I am really stumped on this. I am not a biologist so please excuse me 
for my lack of knowledge as far as the experiments are concerned. I went 
back and reviewed some data with excellent looking dyeswaps and the only 
difference that I know of is that the RNA was company grade, not 
in-house. Could this just be an issue of RNA quality? Anymore suggestions?

Thanks again
LZ

Naomi Altman wrote:

>If these pairs are dye-swaps, then the M-values should be negatively 
>correlated.  In this case, you have a problem (assuming you 
>normalized the data first).
>
>If they are not dye-swaps, then perhaps they are OK.  You cannot pay 
>too much attention to the smooths outside the main body of the data.
>
>--Naomi
>
>At 11:19 AM 2/6/2006, 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.
>>
>>Thanks
>>LZ
>>
>>    
>>
>
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