Dear Bioconductor Users,
I am trying to use LimmaGUI to analyze a CpG island experiment.

I wish to calculate the statistical significance associated with the 
comparison
(Tumor Undigested-Tumor Digested) vs. (Normal Undigested-Normal 
digested).
(Undigested has both methylated and unmethylated CpG islands. Digested 
has only
Unmethylated CpG islands. So Undigested-Digested gives the extent of 
methylation.

Comparing tumor to normal gives the change in methylation associated 
with cancer.

 

Mytarget matrix is:
SlideNumber FileName Cy3 Cy5 Date

18370n_13430283.spot Ndigest Nundigest  10/3/06
28370n_13430572.spot Nundigest  Ndigest 10/3/06
38370t_13469544.spot  Tdigest Tundigest10/17/06
48370t_13469546.spot  TundigestTdigest 10/17/06

 

My problem: I have been able to calculated undigested vs digested for 
normal
and undigested vs digested for tumor, but I have not been able to 
calculate
(Tumor Undigested-Tumor Digested) vs. (Normal Undigested-Normal 
digested)

I first produced a parameterization consisting of
1. (Tumor Undigested-Tumor Digested)
2. (Normal Undigested-Normal digested)

 

And got the following design matrix

  (Nundigest)-(Ndigest) (Tundigest)-(Tdigest)

1	1	0

2	-1 	0

3	0	1

4	0	-1

 

I tried to produce a new parameterization that would calculate

 (Tumor Undigested-Tumor Digested) vs. (Normal Undigested-Normal 
digested)
I edited the design matrix to read:

 

  (Nundigest)-(Ndigest) (Tundigest)-(Tdigest)

1	-1 	1

2	0	0

3	0	0

4	0	0

Is this correct?

 Anyway, it still gave me the same parameters as above:
1.(Tumor Undigested-Tumor Digested)
2.(Normal Undigested-Normal digested)

 

And, when I tried to produce the contrast
(Tumor Undigested-Tumor Digested) -. (Normal Undigested-Normal digested)
I got the following message:

“for this experiment all possible RNA comparisons can be obtained from 
one parameterization. This is because there is an unconnected 
experimental design and there
are only 2 RNA subtypes within each sub-experiment”.

My question:

Is there any way to compute
(Tumor Undigested-Tumor Digested) - (Normal Undigested-Normal digested) 
using
LimmaGUI?

I am using LimmaGUI version 1.3.9 using Limma Version 2,.0.2 under R 
2.1.0 on a PC. It took me some effort to get LimmaGUI working so I 
haven’t upgraded but will if you think it would help., I would prefer 
to remain within the context of LimmaGUI as distinct from Limma if at 
all possible. Also, this is the experimental design that the 
experimentalists handed me. but if it is impossible to use LimmaGI and 
or Limma to analyze this design, please let me know.

Any suggestions would be appreciated.

Thanks as always,
Rich
------------------------------------------------------------
Richard A. Friedman, PhD
Associate Research Scientist
Herbert Irving Comprehensive Cancer Center
Oncoinformatics Core
Lecturer
Department of Biomedical Informatics
Box 95, Room 130BB or P&S 1-420C
Columbia University Medical Center
630 W. 168th St.
New York, NY 10032
(212)305-6901 (5-6901) (voice)
friedman@cancercenter.columbia.edu
http://cancercenter.columbia.edu/~friedman/

"My novel has a hundred characters and it takes place over a year.
When I finish it, if it is shorter than Ulysses, then Joyce described 
things
too much".
-Rose Friedman, age  9


	[[alternative text/enriched version deleted]]

