Normalization is a very basic question each scientist will ask her or himself at the begining of gene expression analysis. Often its importance is ignored or being forgotten because most of the people don;t do that often and there are bodies of papers published lacking proper normalization.
You are right Ahmed.
It is very important to test at least 10 different reference genes then selecting 3 most stable ones using a common protocole like geNorm or geNormPlus. Finally using the geometric mean of the 3 most stable reference gene.
It is just to open a question and find how people are doing that. Opening this question I would like to extend the knowlege, because still there are coming papers in good journals using single reference gene, classical reference genes, not checking for stability of the reference genes etc.
Even if they are done, no information is given within the paper.
I would like to stress out that follwoing MIQE guidlines is vital to be able to repeat mRNA expression profiling studies.
What do you think? what is your experience
But the method you have told.. Is that full proof.... Why Geometric mean and not arithmetic mean?? please elaborate..
By this method you can be sure of the stability.
The reason for geometric means is mentioned in a course by the inventors of the mthod.
vandesopmle Hellemans (Biogazelle):
- controls for outliers
- compensates for differences in expression level between the reference genes
Although I was using the same protocol, you have mentioned, but had no reference. Thank you very much.
Hello everybody,
I am just about to initiate my experiemnts, and I slected 6 reference genes, but I still didn't decide which program I am gonna use for reference gene selection. GeneNorm or GeneNormPlus are licensed, right?! Do you know if there is an opened software that I can use? Thanks ;-)
There is a free license for geNormPlus and old version of geNorm which works on excell 2003 platform. Indeed there are other software i.e. Normfinder, R package programm to test stability and do normalization as well.
I have used, ABI Dataassist. Its a nice tool for a lot many thing with TLDA array data
Thanks Behnam, but which one is friendly user? Navonil, is your software free use?
Behnam, I am trying to download geNormPlus, but at first I need to create a mybiogazelle count, and after that I need to purchase the license. I think this software in no longer opened....
In the same page where you download qBasePlus software, there is a menu named "my licenses". There you can request for a trial license.
To be able to manage large experiments you need to purchase license, otherwise it wont be possible to handle normalization. still reference gene stability check will be possible.
Thank you Philip, I got the paper and I will take a look at the website. I really apreciate the tips. Regards, Aly
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