[BioC] Two populations on microarray

[Gordon K Smyth]

Dear Joaquin,

What I had in mind was that you would make a vector z which takes values 
TRUE or FALSE depending on whether each probe on the array belongs to 
group 1 or group 2 according to your MA plot.  Then

   imageplot(z,layout,low="white",high="blue")

There is no way for you normalize out this problem, and certainly not
within the limited capabilities of GenePix software.

Best wishes
Gordon

---------------------------------------------
Professor Gordon K Smyth,
Bioinformatics Division,
Walter and Eliza Hall Institute of Medical Research,
1G Royal Parade, Parkville, Vic 3052, Australia.
smyth at wehi.edu.au
http://www.wehi.edu.au
http://www.statsci.org/smyth


On Thu, 19 Jan 2012, Joaquin Martinez wrote:

> Dear Naomi, Gordon and Ben,
>
>
>
> Thank you for your replies to Ben Tupper’s (and my) question.
>
>
>
> We are using spotted oligonucleotide microarrays containing probes for both
> host and virus genes. In our experiment we had cultures grown under high
> and low phosphate conditions, inoculated with 2 different viruses
> (separately) or kept virus-free, in triplicate. RNA purified from those
> cultures at different time points was fluorescently labeled (with Cy-dyes)
> and hybridized onto the microarray slides. You can see a flow chart of our
> experimental design here:
>
> http://dl.dropbox.com/u/8433654/design-concept.pdf
>
>
>
> One slide contains 2 samples which had different experimental treatments.
> Each sample was split into 3, labeled (dye swap) and hybridized onto 3
> different microarray slides in combination with another sample to allow
> technical replication.
>
>
>
> I quantified labeling efficiency prior to hybridizing the samples onto the
> microarray slide, for both dyes I got between 30 and 60 dye molecules per
> 1000 nt (what is the range indicated by the manufacturer for good
> labeling). Also we produced FB plots for the green and the red channels,
> both had similar z-range and saturation range, which we interpreted as a
> proof of good labeling (?). See example:
>
> http://dl.dropbox.com/u/8433654/R-G-imageplot.png
>
>
>
> Both MA clusters that we observe contain a mixture of both host and virus
> probes, ruling out that one complete set of probes failed. Naomi mentioned
> that the nondifferentially expressing genes should cluster around M=0, so
> does that mean that the top cluster corresponds to differentially expressed
> genes?
>
>
>
> We used GenePix Pro to scan and analyze the microarrays. Could we use the
> normalization function in the software (normalize the data in each image so
> that the mean of the median of ratios of all features is equal to 1) as an
> alternative to MA? Or would that simply hide the problem? And then do
> normalization between arrays using the quantile method?
>
>
> Thanks,
>
> Joaquin
>
>
>
>>> From: Naomi Altman <naomi at stat.psu.edu>
>>> Date: January 18, 2012 9:56:45 AM EST
>>> To: Gordon K Smyth <smyth at wehi.EDU.AU>, Ben Tupper <btupper at bigelow.org>
>>> Cc: Bioconductor mailing list <bioconductor at r-project.org>
>>> Subject: Re: [BioC] Two populations on microarray
>>>
>>> Dear Ben,
>>> A typical MA plot has most of the points scattered around the line M=0.
>>  Even if you have 2 populations of probes, the nondifferentially expressing
>> genes should be in that central ellipse.  (The lower cluster does look
>> somewhat like the typical MA plot for raw data.)  I suggest that you do
>> separate MA plots for each population of probes, to see if one set of
>> probes failed.  Or, as Gordon suggests, a population for which labelling
>> failed.
>>>
>>> --Naomi
>>>
>>>
>>> At 05:48 PM 1/14/2012, Gordon K Smyth wrote:
>>>> Dear Ben,
>>>>
>>>> Are you saying that you have deliberately designed two different
>> populations of probes onto your arrays?
>>>>
>>>> Your MA-plot suggests that there is substantial body of spots on the
>> array for which the green channel has failed, hence the 45-degree line at
>> the top of the plot.  These dots likely represent spots with a normal red
>> channel value but close to zero for green.  Normally this would have a
>> technical rather than biological cause.  An imageplot may help you identify
>> where the offending spots are on your array.
>>>>
>>>> On the other hand, if you have deliberately spotted your arrays with
>> two quite different populations of probes, then they probably need to be
>> analysed as separate arrays.
>>>>
>>>> Best wishes
>>>> Gordon
>>>>
>>>>> Date: Thu, 12 Jan 2012 14:28:36 -0500
>>>>> From: Ben Tupper <btupper at bigelow.org>
>>>>> To: bioconductor at r-project.org
>>>>> Subject: [BioC] Two populations on microarray
>>>>>
>>>>> Hello,
>>>>>
>>>>> By virtue of experiment design we have two populations to analyze on
>> each of a suite of Genepix microarrays.  You can see an example in an MA
>> plot here (generated using the excellent limma package) :
>>>>>
>>>>>        http://dl.dropbox.com/u/8433654/BE%20T46h%20slide%2052.png
>>>>>
>>>>> We have been following the steps in the limma user guide, and Ben
>> Bolstad's helpful notes http://tinyurl.com/7346mh9 All of the examples we
>> see appear to have just one population to contend with, which gives us an
>> inkling that we are being naive about our analysis.  We suspect that we'll
>> have to separate the two populations before normalization and analysis.
>>  Are there any guides available for managing two populations like this?
>>>>>
>>>>> Thanks!
>>>>> Ben
>>>>>
>>>>>
>>
>>
>

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