Hi everyone:
I am wondering can I use a multicopy gene as the reference gene for qPCR? If not, what are the potential problems involved?
Thank you!
Hi, it really depends on what you are interested in.
For absolute quantification: if the number of gene copies of your target organism is stable and known then it should not be a problem to use it as long as you consider the copy number in your quantification. If it the copy number of the gene per cell is unstable or variating in the cells of interest then this can be problematic and mess up your quantifications when used as reference.
For relative quantification it does not matter so much as you want to see an increase or decrease and compare it to your starting value.
So it really depends on the organism that you are interested in and the kind of experiment you want to do.
Hi, it really depends on what you are interested in.
For absolute quantification: if the number of gene copies of your target organism is stable and known then it should not be a problem to use it as long as you consider the copy number in your quantification. If it the copy number of the gene per cell is unstable or variating in the cells of interest then this can be problematic and mess up your quantifications when used as reference.
For relative quantification it does not matter so much as you want to see an increase or decrease and compare it to your starting value.
So it really depends on the organism that you are interested in and the kind of experiment you want to do.
Hello
Are you talking about gene expression or cell counting? For cell counting Anna Schötta is right.
For gene expression assessments, multi copy genes might have complicated expression patterns or very high level of expression in total. I would avoid a multi copy gene as a reference in gene expression analysis. Instead I recommend using at least 2 reference genes.
Good luck
If the multiple copies are not 100% identical, then you could have different amplification efficiencies. That would invalidate your assay.
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