QRT- PCR - optimal amplicon size (May/25/2005 )
I am going to perfom quantitative RT-PCR using a ABI7700. With the PCR thing comes a primer design program called Primer Express 2.0. Does anyone has any experience how reliable the results of the designed primers are? I am also a little bit confused in terms of the optimal amplicon size. The program recommands an amplicon size of 150bp. But I have read in the literature about amplicon sizes up tol 250 bp. Since I have to construct a standard, I was wondering if 250 bp might be even better in order to distinguish amplicon and standard on a gel later on.
Thanks for every response!
The size of amplicon actually cab be up to 300bp. However, as your size of amplicon increase, you will expect to see a decrease of PCR efficiency, may be 1.9 or 1.6.....
That you have to determine by running a series of 10 fold dilution of your template.
To me, If you are runing a single PCR assay, that easy. If you are runing multiplex, you have to keep both of your amplicon PCR efficiency at the same level. Other wise, quantification of your both amplicon is not compareble.
By the way, I used to keep my amplicon 100 to 150 bp.
I agree with Yong. 100-150 bp is good...try not to go over 150 or your efficiency will go down. You will learn that qPCR is ALL about efficiency...also, I would recommend going for two primers with the same melting temperature, but you probably already know that. The only other thing I would tell you, is that the primer design software is pretty reliable (in my experience) although you would be wise to do a check yourself for primer-dimer potential, and also for 3' T's and that sort of thing...the software is supposed to sort all of that out for you, but sometimes it's not as finicky as it needs to be.
Thank you guys for your suggestions! I think I am ready to tackle qPCR ;-).