How do I interpret multiple Tm peaks for an assay?
Fluidigm Real-Time PCR Analysis software enables users to perform melting curve analysis following real-time qPCR to determine the specificity of the reaction. Only a single peak should be observed for each reaction to indicate that the reaction was specific in amplifying only your gene of interest. Potential causes of dual or multiple Tm peaks include:
- Primer dimers. The problem can be determined by inclusion of a no template control (NTC). If alternative peaks are observed in the NTC at <60 ˚C, then primer dimers are likely the cause.
- High guanine-cytosine content, which can cause a second peak.
- Co-amplification of genomic DNA and cDNA. This can be determined by including a negative reverse transcription control.
- Amplification of off-target amplicons. Run an appropriate positive control whenever possible.