[BioC] External RNA controls on Rat Gene ST 2.0 chip lfc ~ 1 after xps rma??

[Thornton, Matthew]

Hello!

I am processing Affymetrix gene chip Rat Gene 2.0 ST chips with bioconductor package xps using rma normalization. I have included the ExFold ERCC external RNA controls with 2 mixes of different concentrations. I am able to pull out intensities for the ERCC controls at different points along the processing scheme. If I pull the ERCC raw intensities, order them by increasing concentration, and transform both the concentration and intensity by log base 2, I see a nice sigmoid curve that I can fit with a cubic polynomial.

However, when I pull out the ERCC controls after summarization, when I reorder by concentration, and roughly calculate the log-fold change they are all close to 1?? My supposition is that I am overfitting the data with RMA and that I need to find a better normalization scheme. Does anyone have any ideas for different normalization and summarization methods that I should look at? Like iter-PLIER or FARMS or ? Any advice or comments are welcome.

Thanks,

Matt

matthew.thornton at med.usc.edu