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
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
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
>
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
>>
>>
>>
>
>_______________________________________________
>Bioconductor mailing list
>Bioconductor at stat.math.ethz.ch
>https://stat.ethz.ch/mailman/listinfo/bioconductor
>
>
>
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
>>>
>>>
>>
>>_______________________________________________
>>Bioconductor mailing list
>>Bioconductor at stat.math.ethz.ch
>>https://stat.ethz.ch/mailman/listinfo/bioconductor
>>
>>
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
