Hello,
This is my first time to past my help here, after I got lost from a couple of requests.
As suggested by professor Gordon Smyth, I am writing to get some help on LIMMA package while I am trying to analyze my dual-dye microarray data. Before wrote this email I have I tried following LIMMA guide but still have several places unclear with LIMMA for my data.

The first part of my experiment consists of a loop design to compare the gene expression of at different development stages (10DAP, 22DAP and 35DAP, day-after-pollenization) of the same Brassica line. I have dye swap plus two technical replicates, here is the targets file:
FileName

Cy3

Cy5

AT Oligo 02.11.02.176.gpr.fixed

10DAP

22DAP

AT Oligo 02.11.02.177.gpr.fixed

22DAP

10DAP

AT Oligo 02.11.02.178.gpr.fixed

22DAP

10DAP

AT Oligo 02.11.02.179.gpr.fixed

10DAP

22DAP

AT Oligo 02.11.02.180.gpr.fixed

22DAP

35DAP

AT Oligo 02.11.02.181.gpr.fixed

22DAP

35DAP

AT Oligo 02.11.02.182.gpr.fixed

35DAP

22DAP

AT Oligo 02.11.02.183.gpr.fixed

35DAP

22DAP

AT Oligo 02.11.02.184.gpr.fixed

10DAP

35DAP

AT Oligo 02.11.02.185.gpr.fixed

10DAP

35DAP

AT Oligo 02.11.02.186.gpr.fixed

35DAP

10DAP

AT Oligo 02.11.02.187.gpr.fixed

35DAP

10DAP


This experiment is very similar to the design in LIMMA User's Guide section 7.4, except I have technical replicates. From the Guide, should I have to use one sample like "10DAP" as reference, or any sample for a reference?  My goal is to see which genes are differentiated from 10DAP, 22DAP and 35 DAP. How do I get the results of: 1)which genes are consistently up/down-regulated across the 3 stages? 2) which genes are up-down-regulated at each development stage?

The second part of my experiment is
FileName

DPA

Cy3

Cy5

2009-07-10-atq3.7.3.145-15.gpr

15DPA

WT

MUTANT

2009-07-15-atq3.7.3.146-15.gpr

15DPA

MUTANT

WT

2009-07-15-atq3.7.3.147-15.gpr

15DPA

MUTANT

WT

2009-07-15-atq3.7.3.148-15.gpr

15DPA

WT

MUTANT

2009-07-15-atq3.7.3.149-15.gpr

15DPA

WT

MUTANT

2009-07-17-atq3.7.3.151-20.gpr

20DPA

MUTANT

WT

2009-07-17-atq3.7.3.152-20.gpr

20DPA

WT

MUTANT

2009-07-17-atq3.7.3.153-25.gpr

25DPA

MUTANT

WT

2009-07-17-atq3.7.3.154-25.gpr

25DPA

WT

MUTANT

2009-07-17-atq3.7.3.155-10.gpr

10DPA

MUTANT

WT

2009-07-17-atq3.7.3.156-10.gpr

10DPA

WT

MUTANT

2009-07-17-atq3.7.3.157-30.gpr

30DPA

MUTANT

WT

2009-07-17-atq3.7.3.158-30.gpr

30DPA

WT

MUTANT

2009-07-21-atq3.7.3-159-10.gpr

10DPA

MUTANT

WT

2009-07-21-atq3.7.3-160-20.gpr

20DPA

MUTANT

WT

2009-07-21-atq3.7.3-164-25.gpr

25DPA

MUTANT

WT

2009-07-21-atq3.7.3-256-30.gpr

30DPA

MUTANT

WT

2009-07-22-atq3.7.3-115-10.gpr

10DPA

MUTANT

WT

2009-07-22-atq3.7.3-116-20.gpr

20DPA

MUTANT

WT

2009-07-22-atq3.7.3-117-25.gpr

25DPA

MUTANT

WT

2009-07-22-atq3.7.3-118-30.gpr

30DPA

MUTANT

WT

2009-07-22-atq3.7.3-119-10.gpr

10DPA

WT

MUTANT

2009-07-22-atq3.7.3-120-20.gpr

20DPA

WT

MUTANT

2009-07-22-atq3.7.3-124-25.gpr

25DPA

WT

MUTANT

2009-07-22-atq3.7.3-125-30.gpr

30DPA

WT

MUTANT


This is a time course experiment. Again I want to see the expression differentiation across the stage (10, 15 20, 25 and 30DAP) . Can I split the analysis into five sub-groups instead of a whole? If I split the 25 slides into 5 sub-experiments, my feeling is the variance and normalization would be different from each other. Is this correct?  How do I prepare the biolrep as I do not have any biological replicates?

I would appreciate very much if you could give me some suggestions on these questions. Thank you very much for your help!

Sincerely yours,

Yifang Tan



	[[alternative HTML version deleted]]

