Real-Time PCR/Melting Curve Question - (Nov/03/2006 )
I just ran some RNA samples on real-time RT-pcr that have been treated with DNase. anyway, I used a set of beta actin primers and my amplification curves look great, however, the melting curve for my NO-RT control is the same as the melting curve for the regular sample (with RT).
The amplification curves look completely different between the two, but i was wondering if there could still be DNA amplification even if my samples were treated with DNase?
sure. DNAse is a tricky sort of enzyme and is not always terribly reliable; there are many accounts for and against and most seem to be fairly anecdotal
I found an interesting take on this topic on Ambion's website
anyways, the point is, you could still have some DNA in there.
or, one of your other reagents could be contaminated with a bunch of DNA?
third possibility - are you certain it's not primer-dimer? have you run a quick gel to see what you're getting?
Thanks- yeah it could be a primer-dimer...the Tms are a little different, even though the peaks on the melting curve are in the same location. Can primer dimers have a Tm in the 80's? i always thought they show up between 60-7o degrees.
depends on the primer pair
I would certainly rule it out as the first thing