Hi Constanze,
Getting 'AFFX' probes differentially expressed may or may not be a
problem, depending on the probes themselves, so a blanket description
of
'AFFX' isn't helpful.
For instance, probes of the form AFFX-HUM are actually interrogating
the
product of human genes. For instance, beta-Actin, GAPDH, STAT1, etc.
While these are assumed to be 'housekeeping' genes, I don't know that
anybody has shown conclusively that they never change expression from
sample to sample.
The Dap, Phe, Lys, and Thr are bacterial control genes that are added
before the in vitro transcription step as a control to test the
quality
of the IVT step. If they are different, it may signal a problem with
this step. It may also indicate a batch problem, if the chips were run
in batches.
The bioB, bioC, bioD and Cre probes are hybridization controls that
are
added to the fragmented cDNA prior to the hybridization step, and may
indicate differences in the efficacy of the hybridization or batch
problems (e.g., inaccurate pipetting of the controls, differences in
the
hyb oven settings, etc).
If you are getting batch problems that show up in your comparisons,
that
might indicate that the samples weren't randomized to different
batches
(e.g., all the controls were run in one batch, then the experimentals
in
another batch). If that is true then your biological and technical
variability are confounded, and there is no way to know if any
differences are due to underlying biology, or to technical artifact.
One other possibility is that the _5_at and possibly the _M_at probes
are coming up as differentially expressed. All of these probes are
directed towards the 3' end (the _3_at probes), the middle of the
transcript (the _M_at probes) and the 5' end (the _5_at probes).
If you are getting lots of 5' or 5' and middle probes from the
housekeeping genes different in one set of samples vs the other, this
may indicate differential mRNA degradation of the sample. If the same
is
true of the Dap, Phe, Lys, Thr genes, this would indicate a problem
with
the IVT step for some of the samples. An RNA degradation plot would
help
to see if that is a problem for some samples.
Best,
Jim
Constanze Schmitt wrote:
> Hello,
> I am doing a differential expression analysis on Affy HGU95A and
> HGU95AV2 chips. After combining the data into one batch
> (merge.AffyBatch)and jointly normalising with rma (subsequent
quality
> check was ok), I used the "limma" package to fit a linear model
(lmFit)
> and then an empirical Bayes model(eBayes). Results are ranked with
> topTable (fdr correction).
> I get AFFX probes in my top 10 results; as far as I know these
control
> probes should not be differentially expressed. Does this hint at
> possible batch-effects in my data even though there are no outlier
chips
> in the heatmap, intensity- and boxplots of the normalized chips?
> I'd appreciate any help or ideas,
> thanks,
> Constanze
>
>
>
>
>
--
James W. MacDonald, M.S.
Biostatistician
Douglas Lab
University of Michigan
Department of Human Genetics
5912 Buhl
1241 E. Catherine St.
Ann Arbor MI 48109-5618
734-615-7826
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