Hello! I am performing my first qPCR SYBR green assay next week for relative quantification of gene expression of 2 genes + reference gene. I designed and ordered the primers. Their Tm are 56, 57 and 58C. The problem I have is determining the annealing temperature of my qpcr assay since I'm running all samples at the same time. I know I have to do a gradient PCR, my machine has an option for programming a linear gradient. I just don't know how to prepare the samples for the gradient PCR. I know they all have to be identhical, the problem is that I have 3 primer pairs and one cDNA template. Does anyone have a protocol for gradient PCR? Thank you.
You can't use a gradient in your qPCR experiment. You need to use the annealing temperature recommended for your polymerase. Check the product information for your kit.
You should use gradient PCR to see if your primers work well across all temperatures (easy way to check if they are likely to have high enough efficiency) and to make sure you are only amplifying one product. Chances are, you will have to design and test many pairs of primers to find ones that will work.
You have a LOT of testing to do before you can set up the final experiment for this project. As my advisor said "you can't rush quality". But you can waste time and reagents. Have you made your standards for your standard curve?
Good luck!
I usually do gradient PCR using a cheap Taq master mix during the first screening step to see if my primers are good or not. For each primer set, I prepare 8 identical 10-20uL reactions in an 8-strip, and then run a gradient between 55-65C so that each tube in the strip has a slightly different annealing temperature during the run. I do my gradient PCRs to 30 cycles so that I can see if there is an obvious difference in primer performance between the temperatures tested - if you let your reactions go to completion (35-40 cycles) then you won't see any efficiency difference. Running the gradient PCRs on a gel lets you see the approximate temperature range at which the expected product is produced, plus any nonspecific products or primer dimers that form - I have a few primer sets that are dodgy at lower temperatures but perform fine when pushed a little higher, and catching this is the purpose of running a gradient.
With qPCR, you cannot limit the quality of your experiments with statements like 'I'm running all samples at the same time'. That would be nice, sure. But if your primer sets end up requiring different annealing temperatures to work properly, then you simply will not have that option. Your gradient results should let you identify a temperature range of a few degrees where each primer set works, and you will then need to test a few different temperatures on the qPCR instrument to determine the primer efficiency. Remember that for SYBR PCR, equal efficiency of amplification between the reference gene primers and gene-of-interest primers is a core assumption, and without that, your quantification results will be meaningless. Good luck!
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