Hi, If you can get away without pooling, don't. However if you do decide to pool i would use more than two mice per a chip. As another poster mentioned C. M. Kendziorski at university of Wisconsinn has done some really nice work on the effects of pooling in terms of variance components. One key point is that there is the added variance of assigning mice to pools as well as the biological variance between mice. She did some nice work on the number of mice needed per a pool to get the variance to an acceptable level as well. The only real cause for pooling is if you really can't get enough Rna and mice are cheap relative to chips. Nicholas > Date: Mon, 6 Oct 2003 14:27:24 +0100 (BST) > From: Wiesner Vos <vos@stats.ox.ac.uk> > Subject: [BioC] Pooling in microarray studies > To: <bioconductor@stat.math.ethz.ch> > Message-ID: <pine.gso.4.31.0310061400050.3048-100000@markov.stats> > Content-Type: TEXT/PLAIN; charset=US-ASCII > > > 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 > >