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

cstrato cstrato at aon.at
Tue Jun 24 22:14:42 CEST 2014

Dear Matt,

If you want to use a different normalization method, I would suggest to 
try MAS5. Alternatively, you are free to play around with different 

As you can see in my vignette 'xpsPreprocess.pdf' you can do the 
calculation stepwise, i.e. use different methods for background 
correction, normalization and summarization, e.g. I would try sector 
background (mas4), then median normalization and lowess summarization.

For FARMS you can do no bgrd-correction, quantile normalization and 
farms summarization, see chapter 5.5. However, if you think that you may 
be overfitting data I would not use quantile normalization.

With respect to PLIER please see my note in Appendix A.1 of vignette 

Best regards,

On 6/24/14 9:38 PM, Thornton, Matthew wrote:
> 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
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