i am doing RT-PCR using standard curve method to detect the presence of archea in my sample. R^2 value showed 0.45, which was suppose to be 0.9. Also my standard DNA concentrations were in the range between 1.24*10^10 t0 4.94*10^10. with the CT values 32.3 t0 34.91 respectively. Also my unknown samples are amplified in the range of 1*10^6 to 1.4*10^7 with the Ct value 16.42 to 20.15 respectively. But according to the principle CT values are inversely proportional to the copy number. In my case its showing directly proportional. Please can anybody help me to find the reason for this..... ???
Kindly see the results table on page 6 in the attached file for more details.
Thank you so much !!!!
Did you perform efficiency tests with your primers? Because the efficiency shown in your results is really bad, that is probably why your R^2 value is so low (at least for your standards). You should run these test with DNA and test different concentrations of DNA to find the best range for the concentration.
Are you sure that the genes that you are testing as standards have a stable copy number? When you want to calculate the copy number of your GOI you should have about two reference genes to which you can correlate the copy number of your GOI.
A single target molecule should give a Ct value around (more or less) 35.
with 10^10 copies your PCR might be overloaded. Further, the range between 5x10^10 and 1x10^10 is not wide enough.
Use 10^8 as the highest concentration serially and dilute down to 10^2 (at least).
Then look at the plot Ct against log2(dilution factor).
Dear Artur, Hilke and Jochen. Sorry, I checked my research gate late and found your responses to my query.Thanks for your valuable feedback. We are going to repeat the experiment based on your advice. Will revert back with further information. Best wishes, Munawwar
Here I attached the result of latest RT-PCR before following up with your advice.
Results saying standards are undetermined.
What would had gone wrong ?
We serially diluted the standards and loaded too. These standards were in 20, 40, 60 and 80ng concentration.
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