[BioC] Experimental Design Question(s)
Nathan.Watson-Haigh at csiro.au
Nathan.Watson-Haigh at csiro.au
Wed Oct 1 01:50:51 CEST 2008
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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