I would like to compare expression of my gene of interest (GOI) between different parts of my dicot plant (leaves, stems, roots). The plant parts are non-treated only (they are just like calibrator). I don't have treated plant parts and I will not have it. How shold I calculate expression? I have results of efficiency for my GOI and reference (HKG) primers and ct values for non-treated parts of plant. In Pfaffl formula we should substract ct of calibrator from ct of treated sample. But I don't have treated sample. How shold I calculate expression when I don't have treated sample? Should I use different than Pfaffl formula?
since, you are not using any treatments, relative quantitation method of expression analysis will not be applicable. try to analyse it through absolute quantitation method
prepare the standard curve produces a linear association between Ct and initial amounts of total RNA or cDNA from serial diluted standards, allowing the determination of the concentration of unknowns based on their Ct values.(Souaze et al., 1996). (may this method will help for you, hope for the best)
Assuming that you have used the equal quantity of RNA for cDNA synthesis and you have also performed standard calibration curves with primer pairs of each GOI (since you have efficacy data), you can follow the absolute quantification method.
1. Normalize the expression level between and within the samples using reference gene (you termed it as HKG)
2. Calculate the expression of GOI in each sample from standard curve by extrapolating the Ct values obtained after normalization.
This will give you absolute quantification of your GOI I each sample.
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