
Hi,

I'm stuck trying to analyse someone's 2-colour Agilent array data.  I intended to use Limma but can't see how it can be done as I don't think they have the right experimental design. Can someone confirm this for me? I've never had to deal with this problem before as everyone I've helped out has either used a common reference or another standard experimental design!

Main problem (I think): they've not used a common reference and don't have enough replicates.

Two cell-lines:, H1, H2.
Each has been infected with three different viruses (x,y and z say) to give 6 samples: H1x, H2x,  H1y, H2y, H1z, H2z.  As a control they kept uninfected cells, H1C and H2C.
They then sampled each at four different time points: t0, t1, t2 and t3 and ran the following arrays (Red vs Green):

H1x_t0 vs H1C_t0      

H1x_t1 vs H1C_t1
H1x_t2 vs H1C_t2
H1x_t3 vs H1C_t3

H1y_t0 vs H1C_t0


H1y_t1 vs H1C_t1

H1y_t2 vs H1C_t2

H1y_t3 vs H1C_t3

H1z_t0 vs H1C_t0


H1z_t1 vs H1C_t1

H1z_t2 vs H1C_t2

H1z_t3 vs H1C_t3

and the equivalent for the H2 cell line, using H2C as the green channel.  

So for each cell line they have 3 technical reps for each of the control time points but no reps for any of the virus time points.  Obviously, overall they have 2 biological reps for each virus at each time point, but I can't see how to make a suitable design/contrast matrix given the lack of a common reference.

They are interested in virus specific gene regulation (eg. differences in gene expression over t0-t3 with "z" infection but not 
with "y" or "x" infection). I presume they used the controls to remove genes that are found to be highly variable in their cell lines.

Can anyone help? I'm at my wit's end and would really appreciate it! I don't want to tell them I can't help if there is a suitable design matrix that would do the trick.  On the other hand, it would be good to know that the experiment is just badly designed so that they can avoid making the same mistake again in the future.

Many thanks,
Gary Speight


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