I am trying to measure the quantity of 4 variants of a gene. These variant has some common sequence, I designed a primer at common sequence and the other in uncommon sequence, This way I will measure the abundance of different variant and then their relative abundance, Now , during optimizing the real time primer with Sybr Green I got two primer set with different efficiency one is 72% and other is 95%, Now my question is does this (differently efficient primer) affect my absolute quantification.
Yes, the primer efficiency will affect your absolute quantification. Your primers have to have efficiency between 95-105% to be publishable. You can NOT use a primer that has only 72% efficiency. You need to continue optimizing or design new primers.
You can correct for differences in primer efficiency using the Pfaffl equation (once both primer sets have 95-105% efficiency).
Article A new mathematical model for relative quantification in real...
http://toptipbio.com/pfaffl-method-qpcr/
Dear Katie A Burnette,
Among the four variant one is rich with around 80% GC and another is rich with 58% GC , I have designed primers for real time pcr with primer X and Primer 3 online software, but every time I got the same 70 % efficiency. Can you suggest me any solution for this? I have tried DMSO 2.5 to 10% , ethyleneglycol, MgCl2 0.5 to 2mM but no improvement.
Debasish Malik try in a PCR to perform the temperature annealing with the gradient option. Another thing is possible to test is a touchdown.
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