[BioC] question on limma package/PCR validation

[Claus-Dieter Mayer]

Hello,

in my opinion (qRT)PCR can validate a lot of things (is the microarray
probing for the right gene? Is that gene really expressed?...), but it
CANNOT validate
the cutoff unless you increase the sample size! To understand that just
imagine a perfect world in which

a) PCR gave you exactly the same results as the microarray
b) you have the time and money to do a PCR for each sample and  all
genes on the array

In that case you would simply duplicate your data-set and (if you used
limma again) obtain exactly the same p-values and be faced with the same
old problem where to place your cut-off. So if you you get a significant
finding with the PCR for a gene that was significant on the microarray
it still could be a false positive!
Re-analysing the same samples with another technology can only remove
technical variabiltiy not biological variability! If PCR has less
technical variability than the microarray the p-values will tend to be
lower for truly differential genes, but they will not be equal to zero,
so there will still be uncertainty, that can only be reduced by
analysing more samples.

Cheers

Claus

Richard Friedman wrote:
> Dear Mouselly,
>
> 	A B>f 0 is a good place to start.
> It is advisable to spot check genes by PCR to validate the cutoff.
>
> Best wishes,
> Rich
>
> On Jul 18, 2007, at 10:06 AM, el mousselly antra wrote:
>
>   
>> hi,
>>   i have a question on  limma package
>>   when we have the result of tnis pakage and we have p-vlue and the
>>  value of B .
>>   i i want to know on wich value of p we consider that genes are DE ,
>>  we take 0.05 is the threshold or what and i see that when we consider
>>  0.05 as threshold wa have a lot of genes DE.
>>   so, i want some rule (threshold)that will be raisonnable and
>>  efficient to detect gnes DE
>>   thank you
>>   sincerly
>>
>>
>>
>>  		
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>
> ------------------------------------------------------------
> Richard A. Friedman, PhD
> Biomedical Informatics Shared Resource
> Lecturer
> Department of Biomedical Informatics
> Educational Coordinator
> Center for Computational Biology and Bioinformatics
> National Center for Multiscale Analysis of Genomic Networks
> Box 95, Room 130BB or P&S 1-420C
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> http://cancercenter.columbia.edu/~friedman/
>
> In memoriam, John Stewart Williamson
>
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>  
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>   

-- 
***********************************************************************************
Dr Claus-D. Mayer                    | http://www.bioss.ac.uk
Biomathematics & Statistics Scotland | email: claus at bioss.ac.uk
Rowett Research Institute            | Telephone: +44 (0) 1224 716652
Aberdeen AB21 9SB, Scotland, UK.     | Fax: +44 (0) 1224 715349