[BioC] Defining and handling replicates

[Jonathan Arthur]

To swap the order of my original two questions:

Gordon K Smyth wrote:

> Does "drawn at a later time point" mean that the RNA was extracted from the 
> same organisim but at a later time? 

The source of mRNA for the microarrays are plates of bacteria cultured from  clinical samples provided by (human) subjects.

In most cases, one patient => one sample => one culture => one RNA extraction => one microarray. I assume each microarray is a biological replicate grouped by the clinical status of the patient (disease vs control).

In one case, however, one patient => one sample => one culture => one RNA extraction => *two* microarrays. The two arrays were performed several months apart but come from the same RNA extraction (frozen during the interim). I assume these are technical replicates.

In another case, one patient => one sample => *two* cultures made several months apart (sample frozen in interim) => two extractions => two microarrays. Is this a biological or technical replicate? The fact it is from the same patient/sample suggests a technical replicate, but the different culture suggests a biological replicate??


> Have you read the sections on technical replication in the Limma User's Guide?  
> That would be the place to start.

Yes, however I am having difficultly rationalising the section 
on "Two Groups: Affymetrix" with the two on "Technical Replication"

If I treat everything as biological replicates, using a group-means
parameterization, the design I use is:

> design <- cbind(disease=c(1,1,1,1,1,0,0,0,0,0,0,0),control=c(0,0,0,0,0,1,1,1,1,1,1,1))

Presumably, I need to do something like:

corfit <- duplicateCorrelation(eset, design, ndups=1, block=c(???))
fit <- lmFit(eset, design, block=c(???), correlation=corfit$consensus)

checking first to make sure corfit$consensus is positive.

But I am not clear on how to define the block vector?

Thanks for your help.

Jonathan