Copy number of normalizer gene - real time normalization (Oct/29/2010 )
I have few queries can anyone help? I have developed knockouts of fungus for transcription factor genes that regulates sec metabolite pathway.I am planning for real time to quantify the expression of that factor gene in knockouts. I have primers for beta actin to be used as normalizer but I later realised that there are two copies of beta actin in the genome?? Does the copy number of the normalizer affect the final expression data of my target gene (is single copy)??
The main purpose of using housekeeping genes (like actin beta) as normalizers for qprc is to control for the amount of input RNA/cDNA, because they are assumed to be expressed at equal levels.In my opinion, the copy number of a housekeeping gene does not really matter. I wouldn`t even expect the expression of a housekeeping gene with a single copy to be half of the expression of the same housekeeping gene with two copies, because there are many levels in cells to control for mRNA-expression. And even if that was the case, it would not affect your data because the expression of your housekeeping gene would again be the same as long as all the cells you analyze for expression of your target gene have the same copy number.