Hello Alexia,

As you May know, melting temperature is not depends on the level of the expression of gene,

But it depends on the fragment size and GC content of the fragment. I suppose you amplify Short fragment but not the whole gene, right?

Usually, for the gene expression analysis by qRT-PCR the primers are selected to amplify the fragment of 100-250 bp of the gene, but not the whole gene.

Sure, the fragments of different genes can have similar melting temperature, if they have similar fragment size and GC content.

Yes, some times it happens. Don't worry.

Hey Alexia,

You also can compare the melting temperatures of your PCR products using any oligocalculators (e.g., http://mcb.berkeley.edu/labs/krantz/tools/oligocalc.html). If the Tm are similar, everything is fine ;).

If you don't know the sequences of your PCR-products, put your primers to the primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome), probably you will find the sequences for your organism.

But these steps, I think, are redundant. When I use new primers I sequence and BLAST the PCR-product. If the sequence is specific to my species, I just want to have only one peak on the melting Curve (plus a small one for primer dimers can appear). I would not care about similarities between different primer pairs in this case.

Good luck!

This is not a problem. The Tms of RT-qPCR products are certainly not specific for any gene. This Tm is determined by the location of the primers, the length of the product and its sequence composition. The purpose of assessing the melting profile is mainly to assure that there is just ONE melt peak in the RT-qPCR product.