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List:       bioconductor
Subject:    [BioC] Experimental Design Question(s)
From:       <Nathan.Watson-Haigh () csiro ! au>
Date:       2008-09-30 23:50:51
Message-ID: 1DA821E397233B4DBB882ED39162224C564BAD6326 () EXNSW-MBX03 ! nexus ! csiro ! au
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I'm trying to understand and analyse a microarray experiment performed by someone \
else, and I've been reading about experimental design in some books and have a few \
questions.

The experiment involves groups of animals assigned to one of 3 time points (measured \
as the number of days since treatment) and administered with either A or B treatment. \
2 out 3 tissue samples are taken from the groups at their designated time point. This \
is basically to identify expression differences over a time course in response to one \
of the two treatments. In addition a control group is used which received neither A \
or B treatment and sampled at a single time point.

Am I correct in thinking I have an incomplete fractional factorial design since each \
group only has 2 of 3 possible tissue samples taken and it makes no sense to have \
control groups for ever time point and a control can not be treated with either A or \
B? E.g. as mentioned at the bottom of this page: \
http://www.socialresearchmethods.net/kb/expfact2.php

I was thinking of the below as a possible design. '-' no group possible as it's a \
control group

Tissue 1:
     days since treatment
      0    3    7    21
C     8    -    -    -
A     -    4    4    4
B     -    4    4    4

Tissue 2:
     days since treatment
      0    3    7    21
C     4    -    -    -
A     -    4    4    4
B     -    0    0    0

Tissue 3:
     days since treatment
      0    3    7    21
C     4    -    -    -
A     -    0    0    0
B     -    4    4    4

I'm wondering how best to approach the analysis in limma such that the following can \
be answered: 1) Identify genes that change in expression over the days since \
treatment. 2) Genes that show tissue specific responses
3) Genes that show the same response over tissues - I don't think this can be done \
since all three tissue samples were not taken for both treatments, only Tissue 1 was \
taken for both treatments. 4) Find genes in Tissue 1 that respond to both treatments.

I think I'm getting confused with parameterising the experiment into an appropriate \
design matrix. Should I parameterise it using 3 factors (treatment, days since \
treatment and tissue) with 3 (control, A and B), 4 (0, 3, 7 and 21) and 3 (1, 2 and \
                3) levels respectively:
Treatment: Control, A and B
Days since treatment: 0, 3, 7 and 21
Tissue: 1, 2 and 3

Since it's only possible to have days since treatment for non-control groups, how do \
I best approach the analysis? Should I normalise the expression levels to that of the \
corresponding controls for that tissue and then proceed with the analysis using a \
fractional factorial design: Tissue 1:
     days since treatment
      3    7    21
A     4    4    4
B     4    4    4

Tissue 2:
     days since treatment
      3    7    21
A     4    4    4
B     0    0    0

Tissue 3:
     days since treatment
      3    7    21
A     0    0    0
B     4    4    4


Thanks for any input!
Nathan

-------------------------------------------------------------
Dr. Nathan S. Watson-Haigh
OCE Post Doctoral Fellow
CSIRO Livestock Industries
J M Rendel Laboratory
Rockhampton
QLD 4701                              Tel: +61 (0)7 4923 8121
Australia                             Fax: +61 (0)7 4923 8222

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