[BioC] how to handle pooled replicate?

[Jianping Jin]

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.

best,

JP-


--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
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University of Chapel Hill
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Phone: (919)843-6105
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E-Mail: jjin at email.unc.edu