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

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...