It is due to high GC content. But if your primer works, then I think this type of melt curve won't be any problematic.

yes i agree with Maynak Chakraborty ·, because high GC conten   

I would redesign your primers. The suggestion of GC content has merit, but the fact you're seeing this phenomenon stochastically, and in a sample-specific fashion, makes me think this isn't the real answer. Do your primers span introns? Do they also span exons which may or may not be subject to alternative splicing?

If, for example, your primers produce a 180bp amplicon and span a couple of small exons (and some exons can be ridiculously small), one of which is alternately spliced to give you...say, a 168bp amplicon, then you might see exactly this pattern. Unless you were running a high % agarose gel, you might not distinguish the two bands, either.

Either way, it's safest (in terms of experimental accuracy and peace of mind) to simply design more primers. I usually design two or three pairs per gene, since primers are cheap in comparison to multiple qPCRs.

Ok, thank you very much for you answers, special thanks to J.Hildyard.

I was running 2% agarose gel showing expected product, however, two very very similar products are not able to be distinguished. The primers were designed just from mRNA sequence from ncbi database, without knowledge about exon splicing. Well, I realized, there is one thing that I did not write. The organism has not been sequenced yet and we used mRNA sequences from other very very related organism (as it was used in many published papers, so we were inspired by these papers). The other genes are perfect in term of melting curve, just this gene is a bit weird. Is it possible, that the reason may be also the presence of mismatches in the sequences?

In the case, that this amplicon is sequenced and it is the product, what it should be, is it then all right to use these Ct values for expression evaluation?

Anyway, I will design new set of primers to be sure that it is OK. The primers I used are not so good: (Primer3)

OLIGO                start  len      tm     gc%   any    3' seq

LEFT PRIMER    113   20   60.01   60.00  4.00  3.00   

RIGHT PRIMER  325   20   59.97   45.00  4.00  0.00 

PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00

Thank you.

نتقدم بالشكر الجزيل على هذه المعلومات العلمية القـيمة

Now, both samples were sequenced and the result showed the same product without interfering of other sequences in samples. Anyway, I ordered new set of primers, but what could happen there? What do you think?

Thanks a lot.