[BioC] how to handle pooled replicate?

[Sean Davis]

On 8/1/06 12:42 PM, "Jianping Jin" <jjin at email.unc.edu> wrote:

> Dear Sean,
> 
> Thanks for your comments! Are you saying a data set with technical
> replicates only, like this one, is not appropriate for any limma model, or
> even regular t-test? This was the concern I had in my first help request.

The question that one asks with t-tests (or variants) is if the magnitude of
the change (mean) is outside that expected by chance given the variation in
the measurements.  By variation, one usually means "biologic" variation.
Since you have no biologic replicates, you cannot really estimate the
biologic variation, only some form of technical variation, upon which your
t-testing strategy will be based.  It is "valid", but the interpretation is
not the usual based on biologic replicates.

> Actually the lab researchers conducted RT-PCR, in which they used two
> strategies that may improve the uncertainty caused due to lack of
> biological replicates in microarray assay. One was that they used samples
> that were from separate mice relative to ones for microarray. Secondly they
> selected genes with at least 2-fold change in gene expression for PCR
> verification. The results were pretty consistent between microarray and
> RT-PCR. Can genes with more than 2-fold change in expression avoid possible
> dye effect in general?

I would say that dye effect is, in general, not huge in terms of magnitude,
but I would still be concerned about results without validation, but it
seems your lab collaborator is willing to do that.

Sean