Dear All,
I am currently trying to establish a methodology for measuring the amounts of a target gene (at the gDNA level) in my samples using qPCR (on a ABI 7900 machine using TaqMan reagents and primer/FAM-probe chemistry). However I have problems in that the levels of my targhet that I want to differentiate between are very close via qPCR - only across a couple of Ct's.
Therefore does anyone know of an established or reproducible way I can lower the efficiency of the qPCR reaction so that the differencies between samples will be greater? Does the addition of EDTA or alteration in salt concentration within the reaction change the reaction in a reproducible manner? If I do change these will this then invalidate the quantitative aspect of the qPCR?
Any help or advice would be much appreciated.
Many thanks,
Dominic
I doubt that lowering the efficiency would help! If the PCR remains quantitative, then the decrease in efficiency will be exactly the same whatever the amount of target, and the delta Ct will be the same!
This is an inherent problem of real time PCR. This technique is not adequate to reliably measure small differences.
I would suggest southern blot, using serial dilutions of your different samples.
Hi Oliver,
Thanks for the comments, I think you are right with regards to simply lowering the efficency would equally effect all samples and so not change the Ct values.
However I really do not have large amounts of gDNA to play with so I think I would struggle to run a southern blot - what is the minimum amount of gDNA I could use? Is it possible to perform a pre-amplification step and retain quantitative values?
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