limma: can a separate channel microarray analysis done with this setup

Question

[Antonio R. Franco]

A private company has done a microarray bioinformatic analysis with 2 close-related bacteria (TIGR4 and R6) under 2 different conditions (C and DFO)

> targets
  numero FileName      Cy3      Cy5
A      1    A.txt      R6C    R6DFO
B      2    B.txt    R6DFO      R6C
C      3    C.txt TIGR4DFO    R6DFO
D      4    D.txt    R6DFO TIGR4DFO
E      5    E.txt   TIGR4C TIGR4DFO
F      6    F.txt TIGR4DFO   TIGR4C
G      7    G.txt      R6C   TIGR4C
H      8    H.txt   TIGR4C      R6C

They did the limma analysis through a "separate channel" approach.

I am wondering if this "separate channel" analysis can be done with this experimental design or not

I mention this because i did myself a "Direct Two Color" analysis, and the number of DE genes with p<0.05 obtained with the "separate channel" is substantially higher than when the direct design is used

Answer 1

[Charles Warden]

I'm a bit confused with the experimental design. I think samples A, B, E and F make sense, but I'm not sure why they are doing the dye-swaps between species (I would compare DFO vs. C and then look at the differences in differentially expressed genes when defined separately for each species).

What is your goal? Can you visualize the expression of the differentially expressed genes to see if they have expression patterns that look like a reasonable match for the trends that you want to see?

[Antonio R. Franco]

You are right. The design was done in a time where the company was able to convince my colleague to run the experiment this way. These two bacteria share a very high degree of conservation at the genomic sequence

However, my colleague is only interested in comparing the C and DFO conditions within a determined bacteria strain.

I think I will go by analyzing the A, B, E and F arrays through separate channels

[User 59]

Looks like a circular design to me with dye sways?

Answer 2

[Antonio R. Franco]

It is a circular design. The case is the list of DE genes change dramatically when it is analyzed as circular or as separate channels The design is circular, but the company did analyze data as separate channels.