"specific" primers showing 2 dissociation peaks

[mandrita]

Hello All

I have a pair of primers for qRT-PCR. I have designed using a software that uses a mispriming library. I have also blasted the sequences to see if they are specific within all ESTS, and they are. They are also on different exons to not pick up genomic DNA contamination in my cDNA. I also do a DNase digestion (15min, RT) during RNA prep.
However, I am seeing 2 peaks in the dissociations curves of this pair, a sharp big peak and a smaller sharp peak. What can it mean? And is there anything I can do to get rid of it? Can I ignore it?

thanks
mandrita

[subit]

Hi Mandrita,
Well there could be many reasons. First you have to check the temp of the dissociation curve of the smaller peak.Is it much lower than the big peak? If its then one of the reason could be formation of primer dimer.As primer dimer form smaller product so its dissociation temp is lower than the expected product and its very transparent in the dissociation curve.So I will suggest you to run a reaction without template(even without RT) as if primer dimer is forming Taq itself is sufficient.
Second, you can run a reaction with everything without Reverse transcriptase to make sure there is no contamination of DNA. And compare the dissociation curve and the Ct value with one having RT. This can give you idea whether your RNA is really free of DNA contamination and the second dissociation curve is not for that.
Thirdly try to make new primer set or go for fresh RNA extraction having treated with Rnase free DNASE twice each for 15min.
Hope it wil give some breakthrogh
Thanks
Subit

[bharuch]

check if there is a SNP in that location