[BioC] Pooling in microarray studies

[Tan, MinHan]

- 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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