[BioC] RMA normalization,which samples should be normalized together

[Dipl.-Ing. Johannes Rainer]

thanks arne

i have no replicates, affymetrix is still a little bit expensive ;) . 
all our chips were made by ourself and by looking at the histograms of 
the raw values there are no differences at all. in the whole experiment 
we made also two replicates, one with the same RNA, but different 
amount before amplification (one time 5 mug, the second time 1 mug) and 
the second replicate is RNA from the same patient, same time point, but 
the RNA was extracted by two different people  not at the same time. if 
i normalize only those replicated chips i see nearly no differences 
between them (with a M (log2 regulation value) cut off of M=1 i get 
about 30 probe sets that differ), but when i normalize all 80 chips of 
all patients together the replicated chips show more differences... in 
my opinion i have to normalize all patient chips together, exspecially 
if i want to do for example a wilcox between all 0 hour and 6 hours 
chips.
can you tell me a little bit more about the linear model you have used 
to merge the results?

regards, jo


Quoting Arne.Muller at sanofi-aventis.com:

> Dear Johannes,
>
> I've a study with 84 affy chip to characterize a dose effect of a 
> drug. The study was conducted in 3 different laboratories. There are 
> strong differences betweent the laboratories and I've RMA normalized 
> per laboratory and then merged the results in a single linear moel 
> including the laboratory as an additional factor. Maybe you can make 
> the patient or source of RNA a random factor in a mixe effects model 
> - if you've replication per patient.
>
> Just looking at those genes with a significant dose effect I did not 
> find much differences between normalizing all chips together and  
> normalizing per laboratory.
>
> 	regards,
>
> 	Arne
>
>
>> -----Original Message-----
>> From: bioconductor-bounces at stat.math.ethz.ch
>> [mailto:bioconductor-bounces at stat.math.ethz.ch]On Behalf Of Dipl.-Ing.
>> Johannes Rainer
>> Sent: 07 February 2005 10:13
>> To: bioconductor at stat.math.ethz.ch
>> Subject: [BioC] RMA normalization,which samples should be normalized
>> together
>>
>>
>> hi,
>> we are interested in the response of patients to a special treatment,
>> so we have patient samples before and after treatment. i have
>> normalized this samples in different ways using RMA. As RMA tries to
>> detect and correct probe effects by looking at the expresison
>> levels of
>> the probes across all chips it is not surprising that the outcome of
>> the analysis differs depending on which chips i normalize together.
>> It is clear that i have to normalize all patient samples
>> together if i
>> want to compare the expression values of the genes (lets say using
>> statistical tests). i am also analyzing the chips using the 'old
>> fashioned way' by using M and A values and i suppose it is not
>> problematic at all to compare M values of lets say patient 1, 6 hours
>> sample against 0 hours sample with those from patient 2, also 6 hours
>> versus 0 hours where the chips from the two patients were NOT
>> normalized together.
>>
>> -now my question is if someone else has experience in what samples
>> could and should be normalized together with RMA. I saw that ther are
>> (big) differences in the regulation (M) values if i normalize two
>> different patients together compared with the values that i
>> get when i
>> normalize only samples from the same patients together.
>>
>> thanks in advance
>>
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>