Standard Curve Issues with ABI 7500 qPCR - (Aug/13/2006 )
Hi,
I try to set up a qRT PCR Assay on the ABI 7500 system. I use cleaned up PCR product as template to construct my standard curves for relative quantification. The calculated efficiencies for each assay are pretty good range from 94-98%. But the intercept is always low between 12 and 6. I do not understand why and in how I can reach an intercept of 22. I spend plenty of time to optimize primer and template concentration. Has anybody an idea to get a better intercept and on the other hand I do not understand how I can get so good efficiencies anyway. Can I ignore an intercept of 6???????????????????? 
I try to set up a qRT PCR Assay on the ABI 7500 system. I use cleaned up PCR product as template to construct my standard curves for relative quantification. The calculated efficiencies for each assay are pretty good range from 94-98%. But the intercept is always low between 12 and 6. I do not understand why and in how I can reach an intercept of 22. I spend plenty of time to optimize primer and template concentration. Has anybody an idea to get a better intercept and on the other hand I do not understand how I can get so good efficiencies anyway. Can I ignore an intercept of 6????????????????????
I wouldn't care so much about the intercept, because if you download the "Guide to Performing Relative Quantitation of Gene Expression Using Real-Time Quantitative PCR" from the AB website, you will see that they don't consider the intercept value. As far as I understood, the slope value is very important when you do the dCT method, not if you use the absolute quantitation. I hope the guide I suggested can help you, but maybe you read it already...
By LONG HOLDS of short amplicons (at-rich) at elongaton-extension temp and long primers potentially
you can start ISOTERMAL amplification in ONE cycle - priming and elongation can be
performed many times- then efficiency is more than 2
In CLASSICAL - three step PCR - short primers -long amplicons - situation less probable
because priming elongation are temperature separated ( 55 C vs 72 C)
technically it is not very difficult to perform ISOTHERMAL nick-translation of TAQMANs
by using ONE primer ONE taqman ad ONE singlestranded template smile.gif find OPTIMAL conditions
and Taq NICKtransASE smile.gif will start its NATURAL WORK smile.gif without ANY PCR smile.gif
hi to everyone. i have a real problem. i am working with a gene that is 198 bp. i have already done the relative curve with a housekeeping gene and now i want to do the absolute. i have amplified and cleaned my pcr product and done the dilutions for the standard curve. from 1/1000 to 1/100000000 the lines are allright. At higher dilutions the lines seem as if their no diluted and the 1/1000000000000 line is on or before the 1/ 10000000. I've done this at least 10 times with new dilutions and new cleaned pcr product. if anybody has an idea of what is wrong or on how to improve the picture, please send my a messege at antifo@hotmail.com
thans for your time
antifo,
>> At higher dilutions the lines seem as if their no diluted and the 1/1000000000000 line is on or
>> before the 1/ 10000000. I've done this at least 10 times with new dilutions and new cleaned >> pcr product.
Congratulations, you've just discovered the end of the linear range of your qPCR assay!
This is normal. Every real-time assay will have a point at which making the DNA more and more dilute will have no effect on the position of the curves / Ct values (because of primer-dimers usually) and a similar point at which making the DNA more and more concentrated will have no effect (usually PCR inhibition).
Your linear range (where Ct value is proportional to the log of the template concentration) only goes so far. And it's always a good idea to know where your limits are.