Hi, I have some enquiries regarding to miRNA detection using qPCR approach. In my work, I need to detect the presence of a specific miRNA in HSV-1 using RT-qPCR approach. I performed a two-step qPCR reaction using SYBR Green with the same primers that were listed in a paper for cDNA synthesis and qPCR. However, in the melt curve of qPCR, I found that there was non-specific amplification which showed two distinct peaks. I even tried to modify the cDNA synthesis protocol to a pulse-RT reaction but I still faced the same problem. I’m not sure whether this is due to non-specific amplification of the primers or due to no detection of the miRNA that I’m looking for. I’m currently infecting the cells with virus at MOI of 0.05, could low MOI of virus contribute to this problem too?

Does anyone has any suggestions or opinions about this issue .Your help will be very much appreciated.