Wolfgang Huber | 9 Dec 14:31 2012
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Re: Most diff exp genes are up-regulated... can this be true?

Dear Peter

did you already generate a quality report with 'arrayQualityMetrics'?

Also, I assume you (or the biologist who designed the experiment) knows about some controls, for which it is
already known what they should be doing; or is able to test some of the results using an independent assay. 

The dimness of your arrays however suggests that there could be a problem with data quality that is not easy
to fix by data analysis.

	Best wishes
	Wolfgang

Il giorno Dec 7, 2012, alle ore 10:44 AM, Peter Davidsen
<pkdavidsen@...> ha scritto:

> Dear List,
> 
> I'm analysing some one-color microarray data generated using a custom
> designed Agilent array (their 8 x 60K platform).
> When I compare control samples to treated ones most (i.e. >80%) of the
> differentially expressed transcripts are up-regulated. This pronounced
> up-regulation is independent of type of treatment.
> I have never before experienced such a quantitative difference in the
> number of up- and down-regulated transcripts. Furthermore, I have
> tried to analyse relevant datasets in the GEO that mimics my study
> design in terms of treatment regime, duration of treatment ect. These
> analyses--all in closely related species--suggest that the fraction of
> up and down-regulated transcripts should be roughly the same.
> The QC reports generated by the Agilent Feature Extraction Software
> indicate that the data quality should be fine. Also a few basic
> boxplot before and after normalization haven't raised my suspicion. I
> do, however, find that the median signal intensity for each sample is
> significantly lower than what I've seen in the past with the same
> platform (although targeted against another related species).
> I have tried to normalize my data using both quantile and vsn,
> respectively, with similar result. I have also tried to filter my
> dataset using different intensity filters - again with similar result.
> And finally, I also tried using both limma and SAMR for the
> statistics.
> 
> Have anyone by any chance experienced something similar, and how did
> you deal with this issue of many siggenes going in one direction?
> 
> Many thanks,
> Peter
> 
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