I am analyzing an intestinal microbiota of rats by qpcr. I had already done the efficiency of my primers and analyzed samples of DNA extracted from feces and everything went well. Good melt curve and efficiency between 95 and 110%. When using the DNA extracted from the tissue of the large intestine, I am having problems with the melt curve that are strange as in the image, having two peaks, the reaction is the same, the same primer. Do you know what may be happening? If the presence of the mouse's DNA in greater quantity for being tissue may be hindering the reaction? I've tried with 50, 75 and 100ng of final DNA and the same problem remains