hi.....
martin doherty <martin.doherty at="" ...=""> writes:
>
> Hi,
>
> I have Tumour and Normal technical replicates run on both FFPE and
> RNA-Later.
> These are run on X3P arrays. I am interested in comparing the
outcome from
> both
> fixation methods. When I compare the transcripts classed
> as expressed in both RNA-Later and FFPE the ones found
> unique to FFPE is very small (<5%) - if I assume the RNA-Later
> samples exhibit true expression levels this is a good result.
> The intensities/background from both fixation methods is almost
> equal as well.
>
> When I look for differential expression between tumour/normal using
> both fixation methods. I find that almost half of what is
differentially
> expressed in FFPE is not differentially expressed in RNA-Later. This
> disagreement would indicate that the differential expression in FFPE
> is peppered with false-positves or that RNA-Later is not picking up
> differential expression - I'd guess it is the former.
I don't have specific experience with microarrays on this, but have
looked at
differences between these using PCR - see J. Antonov et al, 2005;
Gene Expression Measurements by Quantitative Real-time PCR Depends on
Short
Amplicons and a Proper Normalization; Laboratory Investigation
85:1040-1050.
It would also be my guess that you are getting false positives in the
ffpe
samples, probably from differing efficiency of hybridization in the
different
ffpe samples (again, just a guess). You could try to assess this by
looking at
how variable the different ffpe hybs are (within condition) and
compare that to
the same thing in the rnalater samples.
Best regards, Darlene
>
> The tests for differential expression was done using CyberT, SAMR
and
> Muilttest,
> and a modified t-test. Does any one have any ideas what other
testing
> methods would
> be worth investigating, or have any idea why there is so many false
> positives
> in FFPE?
>
> Thanks,
>
> Martin
>
--
Darlene Goldstein
?cole Polytechnique F?d?rale de Lausanne (EPFL)
Institut de math?matiques
B?timent MA, Station 8 Tel: +41 21 693 5565
CH-1015 Lausanne Fax: +41 21 693 4303
SWITZERLAND
IMHO, it is related to MAQC (microarray quality control) issue. Google
it and you can find many papers these days on this. The simple
approach you can try is using a "mild" fold change plus a t-test, as
suggested by a paper from Nature Biotech, 24, 1162-1169(2006). I tried
that approach in my own project, which does not work very well though.
You can report your result, which will be appreciated. Another
possible approach is using signal2noise, which is similar to t-test
and used in GSEA.
HTH,
weiwei
On 11/20/06, martin doherty <martin.doherty at="" gmail.com=""> wrote:
> Hi,
>
> I have Tumour and Normal technical replicates run on both FFPE and
> RNA-Later.
> These are run on X3P arrays. I am interested in comparing the
outcome from
> both
> fixation methods. When I compare the transcripts classed
> as expressed in both RNA-Later and FFPE the ones found
> unique to FFPE is very small (<5%) - if I assume the RNA-Later
> samples exhibit true expression levels this is a good result.
> The intensities/background from both fixation methods is almost
> equal as well.
>
> When I look for differential expression between tumour/normal using
> both fixation methods. I find that almost half of what is
differentially
> expressed in FFPE is not differentially expressed in RNA-Later. This
> disagreement would indicate that the differential expression in FFPE
> is peppered with false-positves or that RNA-Later is not picking up
> differential expression - I'd guess it is the former.
>
> The tests for differential expression was done using CyberT, SAMR
and
> Muilttest,
> and a modified t-test. Does any one have any ideas what other
testing
> methods would
> be worth investigating, or have any idea why there is so many false
> positives
> in FFPE?
>
>
> Thanks,
>
> Martin
>
> [[alternative HTML version deleted]]
>
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--
Weiwei Shi, Ph.D
Research Scientist
GeneGO, Inc.
"Did you always know?"
"No, I did not. But I believed..."
---Matrix III
