Trembling melting curves in HRM meth assays - (Aug/12/2010 )

Hi evereyone,
We are starting developing HRM (high resolution melting) assays to quantify methylation in a given locus.
Primers were designed with appropriate softwares (MethylPrimer Express, Applied Biosystems) and assays were set according to standanrd guidelines (Wojdacz et al). The used thermocycler is a RotorGene 6000 (Qiagen). We obtain good amplification levels and after dissociation (65ºC to 95ºC ramp 0.2), apparently two differents peaks, corresponding to meth and unmeth products. Our problem is that melting curves appear not smooth with an ugly trembling. We tried to change primer concentration and annealing temperature, which slightly improved the quality of curves, but not significantly.
We will thank any advice or insight about how to fix our curves!

Rocio

Wobbly melting curves usually indicates a heterogeneous methylation state for HRM, or in particular the occurrence of very inefficient bisulphite conversion.

Can you tell us how you converted your DNA and also how you prepared your genomic DNA for conversion?
cheers

Nick

methylnick on Thu Aug 12 21:08:59 2010 said:

Dear Nick, thank you very much for your reply. We started using commercial standards (methylated and unmethylated)DNA already converted, and included some DNA from cell lines converted with Epitect (Qiagen). Finally, we were able to figure out the reason of the strange look of our curves: inadvertently, working solution of primers were dissolved with ddWater instead of TE! And that made all the difference. Hope this can help other begginners!
Cheers
Rocio