Not so Dr. Tulsulkar! The Ct values are based on a number of factors, especially between different genes. Copy number will dictate but one parameter upon which amplification efficiency relies. Your standards are meant to give you information on how to determine copy number from real samples by creating a standard curve for different genes under your qPCR conditions. Two genes with different primers (even with small differences in GC content) will very seldom have identical Ct values even with the same reaction conditions. I would do it again if I were you depending on what downstream normalization you are going to do, just to make sure you interpret proper efficiencies, R2>0.98 (I don't go with anything less than R2=0.9998). The values you have thus far show decreasing consistency with lower concentrations (a common problem). Do the PCRs two more times and take the averages from the most consistent values to form your standard curve. If the standard curve is your primary method of transcript quantification versus relative quantification, run your standard curves on every plate you make and use each specific standard curve as your normalization base to compensate for small differences on inter-plate runs. Another thing, the final 3 data points where you start seeing Ct>30 always becomes a bit questionable, so revalidation is definitely recommended.

Where exactly is the difference between Ct values? Do the replicates differ or do you have differences between wells with the same copy numbers of different standards?

Did you check for distribution over the plate? Sometimes older oder well-used thermal cyclers show issues with ensuring constant temperatures all over the plates, usually the outer wells are affected. When the temperature is not quite exact, there may be huge differences in amplification efficiency.

Thank you Katrin for quick response, my triplicates are constant the CT values differ between two genes of same copy number, technically the ct values should be same if the copy number is same.  top 6X106 then serially  diluted.

Not so Dr. Tulsulkar! The Ct values are based on a number of factors, especially between different genes. Copy number will dictate but one parameter upon which amplification efficiency relies. Your standards are meant to give you information on how to determine copy number from real samples by creating a standard curve for different genes under your qPCR conditions. Two genes with different primers (even with small differences in GC content) will very seldom have identical Ct values even with the same reaction conditions. I would do it again if I were you depending on what downstream normalization you are going to do, just to make sure you interpret proper efficiencies, R2>0.98 (I don't go with anything less than R2=0.9998). The values you have thus far show decreasing consistency with lower concentrations (a common problem). Do the PCRs two more times and take the averages from the most consistent values to form your standard curve. If the standard curve is your primary method of transcript quantification versus relative quantification, run your standard curves on every plate you make and use each specific standard curve as your normalization base to compensate for small differences on inter-plate runs. Another thing, the final 3 data points where you start seeing Ct>30 always becomes a bit questionable, so revalidation is definitely recommended.

I would like to update on this, the reason why I was seeing the differences in the Ct was due to the salt concentration in the plasmid. My team did not order the desalted plasmids.