Question: Question: How does limma derives its logFC value in two colored arrays?

0

9.1 years ago by

Gordon Smyth ♦

**38k**Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia

Gordon Smyth ♦

**38k**wrote:Dear Sunny,
> Date: Mon, 27 Sep 2010 03:14:01 -0400
> From: Sunny Srivastava <research.baba at="" gmail.com="">
> To: bioconductor <bioconductor at="" stat.math.ethz.ch="">
> Subject: [BioC] Question: How does limma derives its logFC value in
> two colored arrays?
>
> Hello Bioconductor Gurus,
>
>
> I have the a data about gene expression from TWO COLORED Agilent
array. I
> wanted to check differential expression between p3 and wild strain
of yeast.
> In one array p3 is colored with Cy5 and wild is colored with Cy3 and
in the
> second array the dyes are swapped. Assuming I have normalized my
data using
> VSN and obtained M values for the two arrays, I now want to use
limma to
> derive the differentially expressed genes.
>
> My model matrix (say design) in this case will be
>
> p3
> 1
> -1
>
> if wild type is the reference.
>
> If my understanding is correct about how limma analyzes differential
> expression, then M value is the dependent variable, sample
annotation
> (whether p3 or wild, provided by design) is the independent
(explanatory)
> variable, and a linear model is fit per gene using the following
equation.
>
>
> lmFit( M , design)
This is all correct.
> As the data per gene is small, it is better to use eBayes method to
obtain
> genewise p-value. But the object obtained from eBayes (say fit3)
doesn't
> contain the value *logFC*.
Yes it does--the logFC is fit3$coefficients. This terminology is
because
the logFCs are estimated as the coefficients of the linear model.
Best wishes
Gordon
> When I use topTable to order the genes, then
> logFC appears.
>
> The concept of logFC is clear to me in case of a Affy single colored
array
> (ie log (Int_trt/ Int_control) ), but somehow I am still confused
how to
> interpret this in two colored arrays.
>
> In my opinion M value (for each array) should represent logFC if
color bias
> is ignored. How does limma derives its logFC value in two colored
arrays? Is
> it based on the B statistics? Please enlighten me !
>
> Thanks in advance for any help.
>
> Best Regards,
> S.
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modified 9.1 years ago
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Sunny Srivastava •

**340**• written 9.1 years ago by Gordon Smyth ♦**38k**