RT-qPCR systems have an inbuilt optimized PCR conditions programmed which actually works with most of the primers used. I use an Applied Biosystem 96 well StepONE Plus Platform for comparative studies. But to my surprise I never had to change the annealing temperature for my primers or rather the entire PCR conditions for that matter. Whether I use primer for Gene X or Y or Z, even for the endogenous housekeeping controls (singleplexing or multiplexing). Hows that possible!!!! Whether the primers which are so specific for their annealing temperature in routine PCR cyclers forgets the same when inside the real time PCR.. LOL!!! Is the principle of Touchdown PCR working in the background?? Insights please??
qRT-PCR, as opposed to conventional PCR, demands that all primers used have the same annealing temperature, because you run 2 or more assays simultaneously (at least one reference and one target gene). None of the qPCR cyclers that I know can carry out two sets of reactions with different cycling parameters at the same time.
So, it is recommended that you design your primers aiming at a constant (usually 60ºC) annealing TºC or buy pre-designed assays.
qRT-PCR, as opposed to conventional PCR, demands that all primers used have the same annealing temperature, because you run 2 or more assays simultaneously (at least one reference and one target gene). None of the qPCR cyclers that I know can carry out two sets of reactions with different cycling parameters at the same time.
So, it is recommended that you design your primers aiming at a constant (usually 60ºC) annealing TºC or buy pre-designed assays.
I worked on a lot of multiplex and yes, it is better to have all the primers working at the same temp. It is a question of efficacy, of your system. But, when you do not have this choice, you have to play with the concentration and the primer design to have good slope (efficiency) and sensitivity. These sides are time consuming, especially if you need sensitivity (low starting copy number: 5-10)
good luck
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