[BioC] Pooling in microarray studies

[Crispin Miller]

Our experience is that it's really important to be consistent - if you use 8ug for one sample use 8ug for all the rest... 
crispin

> -----Original Message-----
> From: Wiesner Vos [mailto:vos at stats.ox.ac.uk]
> Sent: 06 October 2003 15:02
> To: Tan, MinHan
> Cc: bioconductor at stat.math.ethz.ch
> Subject: RE: [BioC] Pooling in microarray studies
> 
> 
> 
> 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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