[BioC] normalisation for universal reference in 2 channel arrays

Gordon Smyth smyth at wehi.edu.au
Thu Jul 29 00:59:56 CEST 2004

At 06:02 AM 29/07/2004, Peter Wilkinson wrote:
>I would like to know some alternative to normalization for 2 channel 
>experiments against universal, where samples may have been hybridized in 
>seperate batches where the universal RNA has changed lot (should not have 
>happened but it did). What is the same between the batches is that what 
>looks like up-regulated compared to the universal iis upr-egulated, and 
>what is down-regulated looks down-regulated. The difference is that the 
>down-regulated (and not in the up), so so much more down-regulated in one 
>of the batches. It looks like to be that the universal has more mRNA 
>abundance in one batch over the other.

Gquantile won't help because it doesn't change the M-values. (It is 
intended for use with single channel analyses.) You could try 'quantile' or 
'scale' normalization (not both) but there are no magic bullets in a 
situation like this. If you use 'scale normalization, you should always do 
within-array normalization first. If you use 'quantile' normalization, the 
within-array normalization step is optional.


>so ...
>I have samples that can be divided into 2 classes: 0,1, and within each 
>class I have samples that have been run at different times. I would like 
>to treat my universal channel uniformly across all samples (assuming that 
>my universal changed lot), and then adjust the Sample (Red) channel to that.
>is the normalizeBetweenArrays with the method="Gquantile" option the right 
>option for this?
>What is the complete work-flow for this case? And after I have normalized 
>within the Arrays, can I go on to scale option for normalizing between arrays.

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