Hi Sean,

 

Thanks much for your tips. On your advice, I did go through the
different examples (designs) in the manual and I am looking for advice
on factorial designs of imagene data.

The data we have is a complex factorial design, which has all the
elements:

Technical replicates, biological replicates, dye-swap design and above
all, it's a factorial design, where both the stimulation(treatment) and
the strain(controls or diabetics) is varied.

I did go through the section (8.7 pg.43) in the limma user's manual on
factorial design, which goes through an example of a single-color array
(affy). What I have is Imagene data (red and green intensities are in
separate files), so I am unable to specify the targets file correctly
and pass the right parameters in 'design' for lmFit to work correctly.

 

Giving you an idea of the design:

 

Each hybridization was between 'stimulated' and 'unstimulated' cells
from the same sample. Hence the 'treatment' was varied on the same slide
(Cy3-stim, Cy5-Unstim and vice-versa). Each strain (control or diabetic)
has two slides, with a dye-flip(Slide1: Cy3-stim, Cy5-Unstim and Slide2:
Cy5-stim, Cy3-Unstim). So there are two technical replicates for each
sample. Further, there are 8 controls and 8 diabetics, so 8 biological
replicates in each group.

 

My specific problem is:

 

a) First of all, can I use limma to analyze Imagene data having
technical replicates, biological replicates and dye-flip in a factorial
design (the manual gives an example of affy data, but I am not sure if
the functions are built to read in imagene data having factorial design
?

 

b) How do I set up the design and the targets file to incorporate all
the diabetics and controls together and find the top DE genes which are
expressed in diabetics compared to controls?

 

Again, you may or may not know answer to all of this, but any tips will
be helpful. Again, I am also posting this to the forum, so will
appreciate tips from other expert users.

 

 

Thanks

Parthav Jailwala

Analyst

McGee Center

Medical College of Wisconsin

 

 


	[[alternative HTML version deleted]]

