I would like to compare expression of my gene of interest (GOI) in non-treated leaves, stems, roots of my plant. In that case I don't have any control sample here and it's not possible to have one for such analysis. Efficiencies of GOI and reference gene primers differs above 5% and the efficiencies are not near to 100%. In that case I can't use delta delta ct method. I could use Pfaffl formula, but I don't have control sample (calibrator). How can I analyze my qPCR data?
Hello Malgorzata Majewska ,
you can compare your gene expression with the expression of housekeeping gene. then you can use the delta ct method. no need for control samples. preferably run all your samples with housekeeping gene primers (actin/EF1alpha) as well as your gene of interest. then you can normalize your expression data with the housekeeping gene by using that formula.
I am agree wit Herath, normalizing data based on house keeping genes with give you idea how gene expression is different among various various parts of same plant and so on.
You use the expression of the housekeeping gene as the reference and use the Delta-Delta Ct method with the Pfaffl correction.
Your efficiencies have to be 95%-105% to be publishable.
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