[BioC] LIMMA - fold change direction?

[Jenny Drnevich]

Hi Glyn,

Your design matrix has the first array of the dye-swap pair as 
positive, which has Day27 in the Cy5. Because the M values you're 
testing are log2(R/G), yes a positive M value means upregulated in 
the Day27 samples. Two other suggestions:

1. If you only have 3 biological replicates, the dye-swaps are 
technical replicates. Your analysis is acting as if you had 6 
biological replicates. See the limma vignette limmaUsersGuide() , 
section 8.2 to how to properly account for technical replicates. They 
have an example almost identical to your experiment (2 bio reps with 
dye-swaps).

2. It would be better to call your samples Day17 and Day27 - if you 
should ever have to use them for column names, R & limma can be funny 
about names that begin with a number.

HTH,
Jenny



At 10:42 AM 7/17/2008, Glyn Bradley wrote:
>Hi all
>
>3 bio replicate and dye swap cDNA array experiment comparing 17day and
>27day samples being analyzed with limma
>targets file:
>"Name"  "FileNameCy3"   "FileNameCy5"   "Cy3"   "Cy5"
>         "1-Cy3-Ac-17d.txt"      "1-Cy5-Ac-27d.txt"      "17day" "27day"
>         "2-Cy3-Ac-27d.txt"      "2-Cy5-Ac-17d.txt"      "27day" "17day"
>         "3-Cy3-Ac-17d.txt"      "3-Cy5-Ac-27d.txt"      "17day" "27day"
>         "4-Cy3-Ac27d.txt"       "4-Cy5-Ac17d.txt"       "27day" "17day"
>         "5-Cy3-Ac17d.txt"       "5-Cy5-Ac27d.txt"       "17day" "27day"
>         "6-Cy3-Ac27d.txt"       "6-Cy5-Ac17d.txt"       "27day" "17day"
>
>so after
>...
>RG <- read.maimages(files, source="imagene")
>MA <- normalizeWithinArrays(RG)
>design=c(1,-1,1,-1,1,-1)
>fit <- lmFit(MA, design)
>.
>what direct are the fold changes I get out in
>does a positive FC mean up regulated in the 27day sample?
>
>Thanks
>
>(ps I would've found it useful if 'coefficients' were labelled logFC
>all the way through. But that might be due to my lack of stats
>knowledge!)
>
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Jenny Drnevich, Ph.D.

Functional Genomics Bioinformatics Specialist
W.M. Keck Center for Comparative and Functional Genomics
Roy J. Carver Biotechnology Center
University of Illinois, Urbana-Champaign

330 ERML
1201 W. Gregory Dr.
Urbana, IL 61801
USA

ph: 217-244-7355
fax: 217-265-5066
e-mail: drnevich at illinois.edu