[BioC] Limma contrasts question

[Daniel Brewer]

James W. MacDonald wrote:
> Hi Dan,
> 
> Daniel Brewer wrote:
>> Hello,
>>
>> I have three types of samples PC3M (the control cell line), Knockdown
>> (PC3M with an siRNA knockdown vector) and Scramble (PC3M with a vector
>> with a scrambled sequence).  I have set up my design matrix like this:
>>
>> design <- modelMatrix(targets,ref="PC3M")
>>
>> As one of the comparisons I would like to compare the knockdown vs the
>> controls.  How would one do this?
>>
>> Is something like this correct?
>>
>> contrast.matrix <- makeContrasts("Knockdown vs Controls"=(2*Knockdown -
>> Scramble)/2)
> 
> I doubt it, but given the amount of information we have here, I can't be
> sure. Please read the posting guide and follow its recommendations. At
> the very least we will need to know what type of arrays you are running
> (I assume spotted arrays of some stripe), and what your targets file
> looks like.
> 
> Best,
> 
> Jim
> 
> 
>>
>> Thanks
>>
>> Dan
>>
> 


Hello,

Apologies for not providing enough information.  It is a two colour
Agilent array.  I was trying to keep the post short, but here is the
full information (note I didn't design the experiment).

Targets file:
SlideNumber	ArrayNumber	FileName	Name	Cy3	Cy5
1	1	Input/1_1.txt	1_1	Scramble	Knockdown
1	2	Input/1_2.txt	1_2	Knockdown	PC3M
1	3	Input/1_3.txt	1_3	PNT2	PC3M
1	4	Input/1_4.txt	1_4	Pooled	PNT2
2	2	Input/2_2.txt	2_2	PC3M	Scramble
2	3	Input/2_3.txt	2_3	PNT2	Scramble
3	1	Input/3_1.txt	3_1	PC3M	Pooled
3	2	Input/3_2.txt	3_2	Pooled	Knockdown
3	3	Input/3_3.txt	3_3	Scramble	Pooled
3	4	Input/3_4.txt	3_4	Knockdown	PNT2

PC3M = the control cell line
Knockdown = PC3M with an siRNA knockdown vector
Scramble = PC3M with a vector with a scrambled sequence
PNT2 = Another cell line (not of interest here)
Pooled = poll of knockdowns before you get specific clone, intermediate
between PCM3 and knockdown - a hetrogenious group (not considered here)

Here is the relevant code (before this the standard steps were used to
give normalised data without any controls):

#Differential analysis
design <- modelMatrix(targets,ref="PC3M")
fit <- lmFit(normNoC, design)
contrast.matrix <- makeContrasts("Knockdown vs Controls"=(2*Knockdown -
Scramble)/2, levels=design)
fit2 <- contrasts.fit(fit, contrast.matrix)
fit3 <- eBayes(fit2))

> design
      Knockdown PNT2 Pooled Scramble
 [1,]         1    0      0       -1
 [2,]        -1    0      0        0
 [3,]         0   -1      0        0
 [4,]         0    1     -1        0
 [5,]         0    0      0        1
 [6,]         0   -1      0        1
 [7,]         0    0      1        0
 [8,]         1    0     -1        0
 [9,]         0    0      1       -1
[10,]        -1    1      0        0

> sessionInfo()
R version 2.7.2 (2008-08-25)
x86_64-pc-linux-gnu

locale:
LC_CTYPE=en_GB.UTF-8;LC_NUMERIC=C;LC_TIME=en_GB.UTF-8;LC_COLLATE=en_GB.UTF-8;LC_MONETARY=C;LC_MESSAGES=en_GB.UTF-8;LC_PAPER=en_GB.UTF-8;LC_NAME=C;LC_ADDRESS=C;LC_TELEPHONE=C;LC_MEASUREMENT=en_GB.UTF-8;LC_IDENTIFICATION=C

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base

other attached packages:
[1] limma_2.14.7

So I would like to compare the two controls (scramble and PNT2) with the
knockdown, but I am not sure how to compare one variable in the model
with two.

I would really appreciate your help with this.

Many thanks

Dan

-- 
**************************************************************
Daniel Brewer, Ph.D.

Institute of Cancer Research
Molecular Carcinogenesis
Email: daniel.brewer at icr.ac.uk
**************************************************************

The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP.

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