It depends on the shape of the melting curve. Run the qPCR reaction on agarose with an appropriate % according to the amplicon size.

If you see one band only, the double peaked melting curve could be a reflection of an amplicon with two low-CG-content regions and the results could definitely be considered reliable.

To be even more sure that the single band is really a single band, run in parallel amplicons for which you know that are slightly shorter and slightly longer (+/- 15bp)  than the amplicon you wonder about. If your resolving is good, you should see the positive controls well separated between each other and your band of interest lying between them.

It depends on the shape of the melting curve. Run the qPCR reaction on agarose with an appropriate % according to the amplicon size.

If you see one band only, the double peaked melting curve could be a reflection of an amplicon with two low-CG-content regions and the results could definitely be considered reliable.

To be even more sure that the single band is really a single band, run in parallel amplicons for which you know that are slightly shorter and slightly longer (+/- 15bp)  than the amplicon you wonder about. If your resolving is good, you should see the positive controls well separated between each other and your band of interest lying between them.

Please check the non template control well melt curve and compare the same with the samples.

The another peak in the melt curve may be of primerdimer (due to excess primer concentration in d reaction)

A couple of questions:  just one primer used?  Or did you mean one primer pair?  Also, you said "few and almost all of the samples" showed a double peak.  Was it just a few samples, or almost all of the samples that showed this?  One possible scenario is that (assuming you used a primer pair; FWD and REV primer), is that you are amplifying 2 splice variants of the same target.  Some of the other possibilities have already been addressed by the other posts above.  Are there known splice variants of your target? Keeping splice variants in mind (when working with eukaryotic samples) is always important.

It depend on shap of melt curve , if the extra peak look like shoulder that mean you have primer dimer and if they look as two peak that mean you have non specific product you can test that by run gel electrophoresis , in first situation you will see only one band but in second you will see two band and that indicate you have not specific primer and you need  a new one.

First, run a gel, like the previous posters adviced. If this gives one band at the expected heigth, you could do a calculation of the expected melting curve. A good website for this is http://www.biophys.uni-duesseldorf.de/html/local/POLAND/poland.html, using the setting for 75mM NaCl. Amplicons that have very different %GC at the 5' and 3' end tend to give two peaks. This is often the case if a primer set spans exons1-2 for RT-PCR.

Two melting curves indicate two bands possibly if you have two perfect curves. Try sugestions above by run it on a gel to confirm. If you see two bands, then you're primer isn't specific. Try to re-design and blast it to your own sequence database in order to verify if they are not match into several sequences.

Definitely after qPCR you have to run the gel.

Since you did not specify the nature of the sample, in theory you may have the case of a difference in the template if you have an isoform or a mutation within your amplicon.

In this sense you may also found in the gel one single band. My advice when you get something you do not expect at the PCR is to Sanger sequencing your product. This will guide you into the right direction for primer-redesigning or PCR optimization.

Hi Hidayah,

Do you mind sharing the image of your melt curve generated on Research Gate?