Hi Wolfgang,

Thanks a lot for your suggestions. I'll try to do the background correction
without negative values. Is that biased background correction method
available in BeadStudio?

Allen

On Jan 3, 2008 6:30 AM, Wolfgang Huber <huber@ebi.ac.uk> wrote:

> Dear Allen,
>
> are you saying that your estimated fold changes are compressed towards 1
> because you did not do background correction?
> There is a reason why background correction is done: to increase
> sensitivity of the analysis. Simply to omit background correction
> because you don't like negative net intensities misses the point, and it
> causes exactly what you describe, namely loss of sensitivity.
>
> There are two approaches to this:
> (i) use a biased background correction method that does not produce
> negative values, then you can log-transform
> (ii) use an unbiased background correction method that can produce
> negative values, then do a glog-transform
> The net effect of these approaches is often quite similar in practice.
>
> I would like to refer to the documentation of, or to the authors of, the
> beadarray and lumi packages regarding the best set of functions to
> perform these (e.g. "vst" is along the lines of (ii)).
>
> Best wishes
>  Wolfgang
>
> PS Allen, Please do consider using more specific (=useful) subject lines.
>
> ------------------------------------------------------------------
> Wolfgang Huber  EBI/EMBL  Cambridge UK  http://www.ebi.ac.uk/huber
>
>
>
> affy snp wrote:
> > Hi Sean,
> >
> > Thanks a lot for pointing out this.
> >
> > We knocked down a gene of our interest so I would at least expect
> > that gene would be shown of down-regulated expression in the test
> > sample, that was what I mean "it should not be the case which no
> > genes were significantly up- or down- regulated".
> >
> > We only have one normal and one test sample in our experiment
> > so I just tried testing the fold change for test samples vs. normal
> > sample at the beginning to get a flavor of how many genes would
> > be affected by the knock-down experiment at the expression level.
> > Of this, the median fold change of expression level of around 46713
> > probes is around 1.
> >
> > That was done for the data which was not processed as the microarray
> > facility did previously. What they did before is to do the background
> > subtraction etc and returned us the data with some negative values.
> > For that set of data, after normalization, they were some genes
> > which were significantly expressed and fit our experimental design.
> >
> > So I am wondering, based on the raw data, did I do sth wrong so that
> > it resulted in the conclusion which I got?
> >
> > Best,
> >     Allen
> >
> > On Jan 2, 2008 1:10 PM, Sean Davis <sdavis2@mail.nih.gov> wrote:
> >
> >>
> >> On Jan 2, 2008 12:31 PM, affy snp <affysnp@gmail.com> wrote:
> >>
> >>> Hello, dear list:
> >>>
> >>> I recently had questions about Illumina data two months ago because of
> >>> a large portion of negative values found. Suggested by some expertises
> >>> here, I requested our microarray facility to re-process the data by
> not
> >>> doing
> >>> any global or background subtraction etc. This way, in deed, there is
> no
> >>> negative values existing. But very surprisingly, by applying two
> >>> packages,
> >>> Lumi and Beadarray respectively, after normalization, the fold change
> >>> between the test and normal samples is around 1. Apparently, it should
> >>> not be this case. Can anybody here offer some possible reasons for
> this?
> >>>
> >> Hi, Allen.
> >>
> >> Do you mean that there are no differentially expressed probes?  What
> test
> >> did you do (limma, t-test, etc.)?  And what information suggests that
> "it
> >> should not be the case"?
> >>
> >> If there are not differentially expressed probes, there could be many
> >> reasons, both biological and technical for this being the case.  You
> will
> >> probably need to dig into the data to check the quality closely.  Also,
> make
> >> sure that sample labels were not switched, etc.  In short, when you get
> a
> >> result that you don't expect, you will need to do some troubleshooting
> and
> >> if you come up empty (no technical explanation), then you
> (unfortunately)
> >> need to explain to your lab collaborators (or yourself) that the
> experiment
> >> did not show differences.
> >>
> >> Hope that helps,
> >> Sean
> >>
> >>
>

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