I am facing a problem in my real time experiment. After standardizing the annealing temperature by semi-quantitative PCR, I did real time for my gene of interest. In one of my samples, I got 2 peaks in the melt curve. Thinking it would be non-specificity, I tried by increasing the temperature further by 3 degrees. But still found 2 peaks in the same sample. I ran the product in a gel and found one blazing band and below it another distinct band. Is it still non-specificity or I am starting to think it could be an isoform? Also, in the same sample, with respect to control, protein expression is increasing whereas when I analyzed my real time, the RNA levels are coming low. Please provide your inputs.
See an image of the melting curve below. The red ones are melt peaks of my gene of interest. Green is of housekeeping gene. There are only 2 samples that show two peaks.