[BioC] M vs M plots- dye effect in dyeswap exp
Naomi Altman
naomi at stat.psu.edu
Wed Feb 8 17:07:45 CET 2006
As usual I hit "send" too quickly. I take back the comment about the
plots of R vs R and G vs G.
--Naomi
At 10:48 AM 2/8/2006, zarzabal wrote:
>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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Naomi S. Altman 814-865-3791 (voice)
Associate Professor
Dept. of Statistics 814-863-7114 (fax)
Penn State University 814-865-1348 (Statistics)
University Park, PA 16802-2111
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