[BioC] two color arrays normalization

[Giusy Della Gatta]

Hi Naomi,

I performed the analysis of my micorarrays,but still I don't manage to revert the channels!
My experiment consisted into infect cells with an adenovirus: an emty one
and an adenovirus expressing for a specific protein.  Then I
treated the same cells with a specific drug or simply with the vehicle (DMSO).
I have 6 microarrays: 3 controls DMSO0-treated and 3 samples drug-treated.
In each microarray the green channel is expressing the levels of infected and treated
cells while the red channel are not infected cells. For all the experiments
I have the same RED CHANNEL reference.
I composed the target file as follows: 

Filename	Cy3	Cy5
Control_1b.txt  ctrl	ref
Control_2b.txt	ctrl	ref
Control_3b.txt	ctrl	ref
dexa_dbz_lt_1.txt	treat	ref
dexa_dbz_lt_2.txt	treat	ref
dexa_dbz_lt_3.txt	treat	ref

and the I used the following script:

>targets=readTargets("target_frame_ltbarc_pgedit.giusy")
>RG<-read.maimages(targets$FileName, source="agilent", ext="txt")
>MA<-MA.RG(RG, bc.method="normexp")
>MA<-normalizeWithinArrays(RG, method="loess")
>design <- modelMatrix(targets, ref="ref")
> design
 ctrl treat
[1,]   -1     0
[2,]   -1     0
[3,]   -1     0
[4,]    0    -1
[5,]    0    -1
[6,]    0    -1
>fit<-lmFit(MA,design)
>cont.matrix<-cbind("ctrl-ref"=c(1,0), "treat-ref"=c(0,1))
>cont.matrix
     ctrl-ref treat-ref
[1,]        1         0
[2,]        0         1

>fit2<-contrasts.fit(fit, cont.matrix)
>d1 <- ebayes(fit2)
>toptable(fit2,adjust="fdr")

I don't know if I am still omitting
something, because I have the positive
controls of this experiment  that 
are going exactly in the opposite way!!

May you can help me?

Thank you in advance!
Giusy


-----Original Message-----
From: Naomi Altman [mailto:naomi at stat.psu.edu]
Sent: Mon 2/9/2009 9:56 PM
To: Giusy Della Gatta; bioconductor at stat.math.ethz.ch
Subject: Re: [BioC] two color arrays normalization
 
If there is no dye-swap, then what do you mean by "swapping of the colors"?

--Naomi

At 07:56 PM 2/9/2009, Giusy Della Gatta wrote:

>Hi everybody,
>
>I am analyzing two color Agilent microarrays
>by using LIMMA package.
>In my specific case the red channel is representing
>"the reference" while the green channel is "the treatment".
>Is it enough to use the Target File composition to specify the name 
>of  the samples
>and their corrispondet channels?  Or I have to use other specific commands
>to specify the "swapping" of the colors?
>
>Thank you in advance!
>Regards
>Giusy
>
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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)
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