Hi all,

I'm working with Agilent single-channel microarray data.

However, it's not clear to me whether raw data should be log2 transformed or not (or if it's intrinsically done "behind the scenes"):

1) In some places (http://matticklab.com/index.php?title=Single_channel_analysis_of_Agilent_microarray_data_with_Limma) the data is not logged at all.

2) Elsewhere (https://stat.ethz.ch/pipermail/bioconductor/2011-August/040543.html) it is said that first quantile normalization is applied to raw intensities and the data has to be (log2)transformed afterwards.

3) On Page 14 (limma's manual) the following paragraphs can be read:

EListRaw. Raw Expression list. A class used to store single-channel raw intensities prior to
normalization. Intensities are unlogged. [...].
EList. Expression list. Contains background corrected and normalized log-intensities.
Usually created from an EListRaw objecting using normalizeBetweenArrays().

Does normalizeBetweenArrays() internally (log2)transform the data? [Therefore nothing should be done to the raw data apart from using it as input for normalizeBetweenArrays()]

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So I don't know what am I supposed to do:

a) y <- normalizeBetweenArrays(y,method="quantile")

y$E <- log2(y$E)

b) y$E <- normalizeBetweenArrays(log2(y$E),method="quantile") 
#Transforming the data inside "normalizeBetweenArrays()"

c) Don't change the raw data at all since normalizeBetweenArrays() internally transforms the data (that's my lecture of what's on page 4 of limma's manual (revised 17 June 2014).
 

Any help would be greatly appreciated.

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> sessionInfo()
R version 3.1.1 (2014-07-10)
Platform: x86_64-w64-mingw32/x64 (64-bit)

locale:
[1] LC_COLLATE=English_United States.1252
[2] LC_CTYPE=English_United States.1252   
[3] LC_MONETARY=English_United States.1252
[4] LC_NUMERIC=C                          
[5] LC_TIME=English_United States.1252    

attached base packages:
[1] parallel  stats     graphics  grDevices utils     datasets  methods  
[8] base     

other attached packages:
[1] gplots_2.14.2        lattice_0.20-29      vsn_3.34.0          
[4] Biobase_2.26.0       BiocGenerics_0.12.0  limma_3.22.1        
[7] BiocInstaller_1.16.0

loaded via a namespace (and not attached):
 [1] affy_1.44.0           affyio_1.34.0         bitops_1.0-6         
 [4] caTools_1.17.1        gdata_2.13.3          grid_3.1.0           
 [7] gtools_3.4.1          KernSmooth_2.23-12    preprocessCore_1.28.0
[10] zlibbioc_1.12.0      

Problem found.

It seems like, at least in the version I have been using (limma_3.22.1), the function "normalizeBetweenArrays()"  uses raw/background corrected intensities as input and, internally, it automatically (log2)transforms the data (at the same time it performs the normalization (i.e. quantile normalization)).

Hope this helps to other people.