ChIP-qPCR shift in melt curve - (Jul/27/2010 )
I am working on ChIP-qPCR protocol. As a control for my experiment I have primers for a promoter region where I know that my transcription factor binds. When I run the qPCR following the ChIP protocol, my WCE and ChIP samples both amplify. However, the melt curve for the ChIP samples is shifted to a lower temperature. This also shows up if I run qPCR for another promoter region, and for an exonic region (supposed to be negative control, but it still amplifies). Does anyone have any ideas what could be happening here?
How much is it shifted and to what temp? I find most real-time melt curves (including from ChIP samples) peak from 75-95'C. If it's below that it's probably primer dimer. Run the real-time samples on a gel and see if it's amplifying a different-sized product or primer dimer. How many cycles do you run the real time for? After enough cycles you'll probably get something to amplify from almost anything, though it could be just primer dimer.
biznatch on Wed Jul 28 15:40:32 2010 said:
How much is it shifted and to what temp? I find most real-time melt curves (including from ChIP samples) peak from 75-95'C. If it's below that it's probably primer dimer. Run the real-time samples on a gel and see if it's amplifying a different-sized product or primer dimer. How many cycles do you run the real time for? After enough cycles you'll probably get something to amplify from almost anything, though it could be just primer dimer.
The melt curves are around 75-76 degrees C. It happened for three different pairs of primers and at cycles below 30, which should be ok.
Couple questions...what is WCE?
Also, do you run a negative template control well in which you just put water? If so, do you get anything there? If so, you may very well have a primer dimer; although primer dimers in wells with a template in it typically show up alongside another bump in the melt curve at the correct temperature.
MM
Mighty Mouse on Fri Jul 30 04:01:35 2010 said:
Couple questions...what is WCE?
Also, do you run a negative template control well in which you just put water? If so, do you get anything there? If so, you may very well have a primer dimer; although primer dimers in wells with a template in it typically show up alongside another bump in the melt curve at the correct temperature.
MM
WCE = Whole-Cell Extract, i.e. chromatin that is not immunoprecipitated. Yes, I do run a NTC with water, and there is no amplification in those wells.