Dear List

I was wondering if someone could give me some advice on the workflow for the R/Bioconductor package aroma.light. I have 12 Exiqon arrays, stained with Cy3 and scanned at 3 different PMT settings of 250, 300 and 350 (75% laser power) using a Genepix 4200A Autoloader. These settings are based on the an initial scan with PMT set to auto which scanned each array at ~350. 
 
I have read the papers by H. Bengtsson et al from 2004 and 2006 describing the scanner offset problem and the solution (as implemented in aroma). I am, however rather naive with microarray data handling and I am unsure however how to proceed in terms of analysis.
 
Specifically, do I need to carry out the calibrateMultiscan.matrix procedure for each triplicate of arrays or can I just proceed to affine normalization? Once I have the normalized data to I back transform this using the backtransformAffine.matrix procedure or do I use the data straight after the nomalization? 
 
After this step I am fairly confident and can use SAM and limma to investigate differential expression. I would, however like to use the information gathered from across the scans to maximise my data collection opportunity

Thanks for any advice.

Iain

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