Silvano Piazza piazza at lncib.it
Fri Mar 25 16:28:30 CET 2005

```Hello to everyones,
the experiments that I have to consider is very simple:

I want  to find significant genes between 2 conditions A and B, but I
have only few experiment so I have to collect both ref versus
conditions (A or B) either dye swap experiment (A versus B and B versus
A)

so targets is
SlideNumber	Cy3	Cy5
array1		ref	A
array2		ref	B
array3		ref	B
array4		ref	B
array5		A	B
array6		B	A

of course array5 and array6 are the dye-swap.

So to design the procedure, I follow the LIMMA user guide (by Gordon
Smith), Chapter 14.5 Weaver Mutant Data.

so
>design <- modelMatrix(targets, ref = "ref")
Found unique target names:
B A ref
>design
A	B
array1    0    1
array2    1    0
array3    1    0
array4    1    0
array5   -1    1
array6    1   -1
>fit <- lmFit(MA,design)
>cont.matrix <- makeContrasts(A.B=A-B,levels=design,weight=MA\$weights)
>fit2 <- contrasts.fit(fit, cont.matrix)
> fit2 <- eBayes(fit2)
....omissis...
M        	A         		t  		P.Value         B
209		3.801460 	6.538782  8.315672 1.0000000 -4.209468
2328	1.184194 	7.343676  6.717978 1.0000000 -4.228492
7877	1.904360 	6.504330  6.114349 1.0000000 -4.239110
27187	-4.0759493.771499 -5.783558 1.0000000 -4.246099
3709	3.434542 3.467492  5.639159 1.0000000 -4.249459
7561	2.002753 5.159913  5.616194 1.0000000 -4.250013
7130	2.580527 3.863867  5.600047 1.0000000 -4.250405
19983 	-2.1176246.836539 -5.567882 1.0000000 -4.251194
So all genes have P.Value equal to 1!!!!!!
in previous posts I read that this happen when you have to consider
multivariate test, which i don't known how to manage..., but anyway

1) Am I doing something wrong in the design?
2) Am I doing something wrong in the subsequent evaluation steps?
Any ideas

Thank you to all

Silvano

Dr.Silvano Piazza
LNCIB,
Area Science Park,