Many peaks=different amplicons

It can be due to:

splice isoforms,

gDNA contamination,

unspecific amplification,

primer dimers/secondary structures

You can load your RT-qPCR product on an agarose gel to find out the size of the different products, this will help you to figure out what is the problem.

Many peaks=different amplicons

It can be due to:

splice isoforms,

gDNA contamination,

unspecific amplification,

primer dimers/secondary structures

You can load your RT-qPCR product on an agarose gel to find out the size of the different products, this will help you to figure out what is the problem.

Hey,

blast the primer sequence. It might be that they bind somewhere else.

I would design a new primer pair (the one you have now might not be a good one (otherwise you would have not that many peaks)).

all the best,

Sebastian

Are your samples prokaryotic or eukaryotic? If eukaryotic, then splice isoforms and gDNA contamination (if your primers are not designed to a genomic intron:exon junction) are among the list of possible other 'problem' peaks in addition to your true peak (as Korcan has stated above along with non-specific amp and primer dimers), but if your samples are prokaryotic, the list of possible other peaks shortens to non-specific amplifications, gDNA contamination, and primer dimers (in addition to your true peak); prokaryotes generally do not have alternative splicing isoforms. And I agree with Sebastian above as well: always BLAST your primer sequences against all available data bases to prove to yourself the absolute fidelity of your primers for your specific target of interest.

With eukaryotic samples - in most cases there are alternate splice forms to contend with. Sometimes amplifying all splice forms is desirable, in other cases, it is not... you should always decide which splice variants are of importance to you (if working with eukaryotic samples) beforehand (especially if your sequence is fully annotated in the data bases as to where alternative splicing occurs). VEGF is a good example of this.

Minor peaks especially at lower end of the temperature spectrum will keep comming but one has to worry when there are more than one major peaks. THere are several reasons as siad above. Beyond these, Heterozygous SNPs in the coding region within the amplified fragment will give you extra peaks close to each other.

Hurul,

If the amplicon is long, say more than 400bp, the parts of it can have the different melting temperatures producing more than one melting peak.

Although the usual suspects are isoforms, contaminations, primer dimers and non-specific amplification as already was pointed out by the previous replies.

As already written different melting curves mean that there are different products within the sample. The melting curve describes only the calculated turning point of the fluorescence signal in the well. Hence, if there is only one main product it describes mainly the temperature where half of your dye is already removed from the PCR-product or the other way around when your DNA is opened up to 50%. If there are more products you´ll get more turning points. From my experience SNPs are not so easy to distinguish from the main product if you hadn´t performed a high resolution melting curve because the differences are very minor. Often the peak is then only a bit wider.

In your case I would first of all use these amplified products, like written already, for agarose gel electrophoresis and see if there are more than one product if it is the case direct sequencing or cloning into a vector (then sequencing) would answer the question what kind of further products were produced. You can check your primer pairs as well in a so called in silico PCR against classical genomes (attention normally against DNA so with the introns) to see if there is for example a gene duplication which gives you further products. Contamination with gDNA is every time a problem but can be reduced by good primer design over introns. Primer dimers will have a lower melting temperature compared to your product and will be seen in your gel as very small band.

Thank you all., I've been proceed my sample for sequencing., Then there is my sequence in th sample. Somehow, it's not specific since the template match only for 30bp.

Anyhow, I had been suggested to proceed the expression. Nad the result shows is quite good.

Just to add to Oleg Suslov's answer

Multiple peaks (I personally have seen 2) could result from a product which has a peculiar distribution of GC content.

-Example: Suppose one end of the amplicon has 70% GC and the other has only 40% GC, both the ends of DNA will melt at slightly different temperatures giving 2 peaks.

There are programs that use thermodynamic calculations to predict a melt curve for you. Check uMelt (https://www.dna.utah.edu/umelt/umelt.html)

See example screenshot attached here, the DNA strand is shown in the bottom right