hi guys, i was doing this real time pcr job, in order to determine the regulation of gene expression of mutant and wild type mouse liver cell by the different expression level of the mutant and wild type dna.
from the dissociation curve that i got from the results session, i actually manage to get 2 peaks instead of one. this means that there are 2 types of molecule which dissociate at different temperature, which is an error.
i was wondering if any of you guys know the possible explanation to this? what exactly is the error that i have commited? -candyass-
i get this with some primer sets as well. have you tried running your samples out on a gel? which samples are they that you have the unexpected product in? i find that it's usually my negative reverse transcriptase samples (but sometimes in all my samples) that have these bands in them. This is because when there's no cDNA around the primers can form primer dimers, which then fluoresce when using dyes like Sybr green (is this what you use?)
i find that optimising the primer concentrations sometimes helps. just try adding less primer? or maybe design new primers with low secondary structures, etc.
but if this is what's causing the problem, then it shouldnt affect your results too much. mainly because primer dimers only usually appear in later stages of the PCR - there's usually not enough of then to fluoresce above background until you've done heaps of cycles. so they shouldnt affect your C(t) reading.
hope this helps -ros-
Hi!
I hate to be a PIA, but I disagree with the last part of Ros' answer. If your second peak in the dissociation curve is very large at all (note derivative value and compare to regular expected amplicon peak) then it is NOT showing up too late in the cycles to make a difference in your results. I agree that optimizing primer concentration, in particular trying lower amounts of primers (think a range of 50 to 500 nM) is a great way to solve this problem. I have also found that too little template gives the same result. If your added template amount is too low, or if the gene of interest is expressed at an extremely low rate in comparison with your endogenous reference, then the primer-dimer formation reaction can actually compete with the amplification reaction and this is what you will see.
Good luck!! I hope you are able to get rid of your extra peaks... -aimikins-
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