Hello ResearchGate community,
I have run standard curves on cDNA synthesised from whole mouse liver RNA and for two of my genes of interest I have gotten melt curves that have a smaller secondary peak (see attached images 1 and 2). I am using a SYBR Green Master Mix and running the samples on a Quantstudio 12k flex machine - the rest of my genes of interest appear to be working well and producing a single clean peak (see attached image 3). I am unsure of whether this indicates primer dimerization, non-specific amplification, or is of no concern as I cannot find anything online about what a peak after the primary amplicon peak means... Please help!
Thank you for your time.
Hi Caitlin McCaffrey
Compare the melt curves (images 1 and 2) with the respective negative control melt curves. If the second peak is the same as the negative control peak, it could be due to primer dimer. If not, it's a non-specific amplification.
Hi Govindkumar Balagannavar thank you so much for your response. When you say negative control what are you referring to? I have a no template control (PCR grade H2O with the primers and the SYBR master mix), a no reverse transcriptase control with the primers and SYBR, and then an endogenous control Gapdh. Are you potentially referring to a negative control being a cell type that is known to not express the gene of interest?
Govindkumar Balagannavar Okay so just to check that my understanding is correct, if I were to see a peak between 85 and 90 on the below curve (No template control) that would indicate that it is a primer dimer but because I see no amplification peaks it's more than likely a non-specific product?
I am currently setting up a gel electrophoresis to check if I get more than one band as a secondary confirmation.
Yes, there is no peak at 85–90, hence, it might be non-specific amplification.
Dear McCaffrey,
1. Have you run the agarose gel? I recommend you run the agarose gel to see if there are any other no-specific products.
2. You can also check the melting curve and gel product for no template control to see if that has the same pattern.
3. According to the second melting peak it does not seem to be primer dimer. Primer dimer melting temperature is generally smaller. As the second melting peak is around 90c it could be a non-specific product or may be bacterial contamination in the reagents. If it is a non-specific product then you can try to reduce the PCR cycle numbers to see if this can eliminate the second melting peak.
Best,
Monzur
Thank you so much Syed Monzur Morshed I did do an agarose gel (see below) and could see no obvious non-specific binding by either of the genes of interest and as shown previously above in the string of this question, no peaks present at the same temperatures in the no template controls so I don't think it's a primer dimer either. Still not sure what could have caused it, but I feel confident enough to continue using these primers knowing that it's not dimerization or non-specific binding.
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