[BioC] 2-colour bias AFTER normalising with Limma, labelling QC

[Matthew Hannah]

Hi,

Hopefully someone can help me with this one. I'm fairly sure what might be happening but would like confirmation before I pass judgement to the experimenter. This is basically linked to my earlier post where I was looking at scatterplots to assess reproducibility between replicates.

Anyway, I've move backwards towards the raw data, and I think I have an idea of what is happening. I've attached some files again, they're scrubbed from the email, but you can find them on the html version of the list
https://www.stat.math.ethz.ch/pipermail/bioconductor/2004-July/date.html

The first is the density plot of the Log'd raw intensities. It shows 6 chips. All the red channel intensities peak around 12, the green at 15. All the green channels have a low peak with an extended shoulder to the right. chips 1-3 of the red channel have a similar pattern to the green, whilst 4-6 have more of a spike at 12 and less of a shoulder (4-6 look more like the example in the limma guide!).

If you do a MA plot of any of chips 1-3 (+/- BG correct x +/- normalisewithinarrays), then the MA plot has a 'finger' coming out upwards (5° from horizontal) (see 2nd attach). The BG images of at least 2 of the 3 chips look fine). I think this could be a labelling problem, but still have some questions.
Why is is only at higher intensities?
Is the red the bad label, or (if unlabelled DNA is removed? and taking into account the competitive mixing?) is it actually the green label that is bad?
(I just look at the data) But how do people usually QC the labelling and ensure that exactly the same amount of the 2 samples are mixed?

Thanks in advance,
Matt
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