[BioC] About between-array normalization in limma

[Sean Davis]

De-Jian,ZHAO wrote:
> Hi,everyone.
> 
> I am now analyzing my microarray data using limma. I am not sure
> when to use between-array normalization.
> 
> In the first case of chapter 11 in limma user's guide, it is
> mentioned that between-array normalization is not routine and should
> only be done when there is good evidence. I wonder what is a good
> evidence and whether I should carry out between-array normalization
> in my data. See the attachment for the boxplot.
> 
> In the attached boxplot,the blue, red, and green ones stand for
> three different hybridization groups. There are 6 replications in
> each group. I want to carry out between-array normalization in
> the three groups respectively and then combine them together to fit
> a linear model. Is this reasonable? In my opinion, we can always
> carry out between-array normalization between the replications of
> the same hybridization group (unless there is dye swap).

The rule-of-thumb is that if you are working with two-color data and
using ratios only, then you probably do not need between-array
normalization.  If you want to do single-channel analyses of two-color
data or are using single-channel data (affy, etc.), then you need to use
between-array normalization.

The other rule-of-thumb with normalization is to do the least reasonable
amount possible.  So, despite the fact that one _can_ do between-array
normalization, one should not do it unless there is a good evidence that
it is important, after looking at within-array normalization results.

Sean