[BioC] ttest or fold change

Ramon Diaz-Uriarte rdiaz at cnio.es
Mon Dec 15 12:54:23 MET 2003

Dear Jason,

First, I think you should recognize that three replicates are very few and 
thus conclusions will not be particularly trustworthy. I assume this is a 
first round of screening for relevant genes for subsequent studies. Second, I 
think the fold-ratio vs. t-test issue can often muddle two different 
questions: a) is there statistical evidence of differential expression; b) is 
the expression of gene X altered in a biologically relevant way (where 
biologically relevant means more than Z times). If you had a large number of 
samples you might be able to detect as "statistically significant" very small 
log ratio changes (which might, or might not, be biologically relevant); 
converseley, what if the fold change is large but the variance is huge? For 
reasons I don't understand, the two-fold change sometimes has a sacrosant 
status, but it is my understanding that other fold changes (say 1.3 or 3.5) 
could, on certain cases, be much more biologically relevant; this, of course, 
depends on the context.

In your case, the t-test has an additional potential problem with the 
denominator. I would suggest using some procedure, such as the empirical 
bayes one in limma, that will use a modificied expression for the 
denominator, and save you from  finding some very small p-values just because 
that gene has, by chance, an artificially small variance.

So I would use limma (or something like it) and also filter by some criterion 
that biologists tell you is relevant for them (say, we only want genes that 
are overexpressed at least 5 times, or whatever). 



On Saturday 13 December 2003 16:45, Jason Hipp wrote:
> I am comparinga relatively homogeneous cell culture to another that has
> been treated, and am using RMA.
> I only have 3 replicates of each.  Would you recommend a 2 tailed equal
> variance t test? I also thought I read that with such few replicates, a
> fold change would be better than a t test? If I get a t test of .0001, and
> a fold change of 1.2, is this a reliable change using RMA?
> Thanks,
> Jason
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Ramón Díaz-Uriarte
Bioinformatics Unit
Centro Nacional de Investigaciones Oncológicas (CNIO)
(Spanish National Cancer Center)
Melchor Fernández Almagro, 3
28029 Madrid (Spain)
Fax: +-34-91-224-6972
Phone: +-34-91-224-6900

PGP KeyID: 0xE89B3462

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