[BioC] how to handle pooled replicate?

[Sean Davis]

On 8/1/06 11:40 AM, "Jianping Jin" <jjin at unc.edu> wrote:

> Dear Sean,
> 
> Thanks for your reply! I double checked with the lab researcher about the
> sample pooling. As I understood, the total
> RNA was pooled from 3 mice (wt or ko) and then split into 3 aliquots. Each
> aliquot was separately reverse transcripted and labeled. Two aliquots of
> the labeled cDNAs from wt and ko separately were then mixed, purified and
> hybridized onto an Agilent chip. Hope this is clearer.

So, if I understand correctly, these are not really biologic replicates.
But for the purposes of analysis, they all have the same variance structure
(whatever that is), so can be treated on "equal footing" as far as analysis
is concerned.  Since you don't have biological replication, whatever you
find will be of limited biologic generality.  In other words, if one runs
the experiment again using different mice, the genes that you get may be
different.  

As I mentioned before, the lack of dye swaps is more problematic, as any
differentially expressed gene (if you find any) will be due to EITHER dye
bias or biologic effect.  If you have more than one probe per gene (and for
some genes, that will be the case), and all probes show the same magnitude
and direction of change, that is probably believably not due entirely to dye
effect.  However, there is no way to know for sure and most genes will not
have two or more probes that worked for each gene (on a 44k Agilent array,
at least).  For "publication" purposes you will basically have to run dye
swaps for such a direct design (unless there is going to be validation using
a second technology such as PCR).

Of course, there may be other opinions here, and the data can be used for
many purposes besides strictly "publication", so you will need to make up
your own mind in consultation with the lab researcher.

Sean

> 
> --On Monday, July 31, 2006 2:55 PM -0400 Sean Davis <sdavis2 at MAIL.NIH.GOV>
> wrote:
> 
>> 
>> 
>> 
>> On 7/31/06 2:49 PM, "Jianping Jin" <jjin at email.unc.edu> wrote:
>> 
>>> 
>>> Dear list:
>>> 
>>> There is a data set, consisting of 3 Agilent slides. The experiment was
>>> run with direct hybridization, knock-out versus wild-type, and no dye
>>> swap. Due to difficulty of collecting samples, the samples were pooled
>>> and hybridized onto 3 separate slides.
>> 
>> How were the samples pooled?  Were they pooled and then split, or are
>> there three distinct biologic replicates?
>> 
>> The lack of dye swap IS a problem, as you will likely find dye-biased
>> probes (potentially MANY).
>> 
>>> Of course the 3 slides are not biological replicates. They are not pure
>>> technical replicates either. How should I set up a design matrix for
>>> limma model analysis?
>> 
>> You'll need to be a bit more specific about how you did the pooling....
>> 
>> Sean
>> 
> 
> 
> 
> ##################################
> Jianping Jin Ph.D.
> Bioinformatics scientist
> Center for Bioinformatics
> Room 3133 Bioinformatics building
> CB# 7104, Campus
> Phone: (919)843-6105
> FAX:   (919)843-3103
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