[BioC] is-normalisation-really-required

Thu May 12 04:53:18 CEST 2005

I would add that the reason the TopTable results do not agree with the 
2-fold or more results, is that generally statistical tests compare 
treatment mean differences
to within treatment variation.  Hence, if the results are not variable, you 
will have many statistically significant genes that have less than 2-fold 
difference.  As mentioned many times on this list, statistical significance 
does not imply biological significance, but differences that are not 
statistically significant may be due to chance variation and thus are 
unlikely to have biological significance.  The converse side of this is 
that if genes are highly variable, they may have more than 2-fold 
difference and not be statistically significant.

The purpose of normalization is to remove biases that differ from array to 
array due to the hybridization and labeling processes, so that comparisons 
between conditions are free of this part of the experimental error.  This 
improves our power to detect statistically significant differential expression.

--Naomi

At 09:59 AM 4/13/2005, Gordon Barr wrote:
>Gorjanc and Vijay
>
>This is a misconception as to why to normalize the data. It is not so
>that we can get "pleasing" results or agreement between analytic
>methods but because statistically it is the correct thing to do. If I
>use the wrong statistical test on a set of data (e.g. parametric tests
>on data  that violates all the assumptions) and it gives the same
>result as an appropriate non-parametric analysis that does not make it
>"right" and ok to do again. It means I got lucky. If the analysis of
>non-normalized data is the same as of normalized data you are lucky not
>right. Sean is on target- if they agree normalize; if they do not agree
>normalize. I would add to that why bother analyzing the non-normalized
>data.
>
>Gordon
>
>
>Gordon A. Barr, Ph.D.
>Senior Research Scientist
>NYS Psychiatric Institute
>Columbia College of Physicians and Surgeons
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>On Apr 13, 2005, at 8:29 AM, Gorjanc Gregor wrote:
>
>>>-----Original Message-----
>>>From: Sean Davis [mailto:sdavis2 at mail.nih.gov]
>>>Sent: sre 2005-04-13 14:16
>>>To: Gorjanc Gregor
>>>Cc: bioconductor at stat.math.ethz.ch
>>>Subject: Re: [BioC] is-normalisation-really-required
>>>
>>>On Apr 13, 2005, at 8:03 AM, Gorjanc Gregor wrote:
>>>
>>>>Hi!
>>>>
>>>>You might try analysis with and without normalization and take
>>>>a look at the results. If they say the same thing than I would
>>>>say, no it is not necessary to do normalization.
>>>
>>>So, if the two results agree, then the results with normalization are
>>>correct; if not then the results with normalization are still correct.
>>>Sounds like we are pretty much stuck with normalization....
>>>
>>>Sean
>>Why should one do normalization if the results aren't different. But,
>>in
>>that case it really does not matter and one can do it or not.
>>
>>
>>>>dear friends
>>>>i have situation, where i thought its ok for me not to
>>>>do normalisation, i am afraid i may be wrong. i want
>>>>your advice in this regard.
>>>>
>>>>we performed a wild type - mutant, dye-swap
>>>>experiment.
>>>>when we analysed the intensity values, they were
>>>>consistant among the two experiment (dye-swap). ie.,
>>>>almost same values for mutants in both the experiments
>>>>of the dye-swap.
>>>>since the values are almost same, i thought there
>>>>might not be any dye-bias, so i just went ahead,
>>>>averaged the two values, found out their ratio and
>>>>filtered genes with 2 fold change.
>>>>
>>>>so i have done this without normalisation.
>>>>i am afraid, i might be wrong, my 2 fold chaging genes
>>>>might be wrong...
>>>>kindly give me your advice in this regard.
>>>>i did analyse the data with limma, but the topTable
>>>>genes there never correlates with my 2 fold genes.
>>>>
>>>>kindly correct me.
>>>>thanks
>>>>
>>>>vijay
>>>>graduate student
>>>>department of biological sciences
>>>>the university of southern mississippi
>>>>MS, USA
>>>
>>>--
>>>Lep pozdrav / With regards,
>>>     Gregor Gorjanc
>>>
>>>---------------------------------------------------------------------- -
>>>-
>>>University of Ljubljana
>>>Biotechnical Faculty       URI: http://www.bfro.uni-lj.si/MR/ggorjan
>>>Zootechnical Department    email: gregor.gorjanc <at> bfro.uni-lj.si
>>>Groblje 3                  tel: +386 (0)1 72 17 861
>>>SI-1230 Domzale            fax: +386 (0)1 72 17 888
>>>Slovenia
>>>
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Naomi S. Altman                                814-865-3791 (voice)
Associate Professor
Bioinformatics Consulting Center
Dept. of Statistics                              814-863-7114 (fax)
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