[BioC] Pooling in microarray studies

[Wiesner Vos]

Thanks for the replies Min-han and Jim.
I suspected that this may be the case (keeping the
papers in mind about comparing expression measures
on the Dilution data eg. Cope et al.)

I suspected that the use 8ug would be perhaps
not make much of a difference if you use
an expression measure like for example
RMA that seems to be reasonably independent of
the actual amount of RNA on a chip. Thanks
again for your help.



Wiesner J. Vos
Department of Statistics
University of Oxford
1 South Parks Road
OX1 3TG
United Kingdom






On Mon, 6 Oct 2003, Tan, MinHan wrote:

> - Perhaps it is an policy issue with your microarray core facility, but
> the standard Affymetrix GeneChip Technical Manual recommends 5 - 20 ug
> total RNA.
>
> - I routinely use 5 ug total RNA with no apparent problems.
>
> - So yes, you should be able to do single sample hybridizations.
>
> Regards,
> Min-Han
>
>
>
> -----Original Message-----
> From: Wiesner Vos [mailto:vos at stats.ox.ac.uk]
> Sent: Monday, October 06, 2003 9:27 AM
> To: bioconductor at stat.math.ethz.ch
> Subject: [BioC] Pooling in microarray studies
>
>
>
> I have question arising to the pooling of mRNA
> samples. Someone approached me about the
> following problem:
>
> The study wants to use Affymetrix chips to study
> changes in expression between a group of treated
> mice and a group untreated mice. There are 10 mice
> in each group. It is only possible to extract
> 8 ug of RNA from each mouse, not enough for one chip. (According to the
> experimenters they require 10 ug per
> chip)  So it is not possible to use biological
> replicate chips for each individual mice. Now the issue
> is whether to perhaps pool the RNA in each group
> and carry out analysis on technical replicates from the
> pooled samples.
>
> As I understand it pooling may reduce the precision, with
> the risk that one or few samples can dominate the outcome, and that
> averaging over single sample hybridisations is perhaps safer than using
> pooled samples. However in this case you cannot do single sample
> hybridisations.
>
> I was wondering if the following approach is an acceptable compromise to
> retain at least some information on the between sample variation in each
> group:
>
> Mix the RNA from 2 different mice on a single chip to get 5
> hybridisations, where the hybridisation on each chip is from the mix of
> the RNA samples of two mice? I though that this may enable you to some
> extend if all the mice are behaving similarly. Ofcourse one would not be
> able to distinguish between the behaviour of the two mice relating to
> the same chip. Or is it better to accept that you do not have enough RNA
> to hybridize the sample for each individual to a separate chip and pool
> the samples and accept the risk that one sample may dominate the
> outcome? The best solution did not seem obvious (to me at least!)
>
> Any comments will be much appreciated.
>
> Wiesner
>
>
>
> Wiesner J. Vos
> Department of Statistics
> University of Oxford
> 1 South Parks Road
> OX1 3TG
> United Kingdom
>
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