I have selected a set of primer sequences for my RT-PCR experiments, using either the NCBI Primer Blast software or Primer3. To ensure the specificity of these primers before ordering, I employed in silico design tools and NCBI Primer Blast. Some of the sequences showed 100% specificity, as they produced the same protein name, product size, and melting temperature (Tm). However, upon testing their specificity, some of them yielded different product sizes and occasionally showed associations with other proteins that are not of interest to our study. I referred to certain publications where our genes of interest were investigated, and when I tested the specificity of the primer sequences used in those studies, I encountered the same issue. For example, in human cells, Ezrin was studied using the following primer sequences: Ezrin-forward: 5′-CGCTCTAAGGTTCTGCTCT-3′, Ezrin-reverse: 5′-TCCTGGGCAGACACCTTCTTA-3′. However, when I tested the specificity of these primers, the name of the resulting gene product was different from Ezrin (as shown in the image), and the product size exceeded 3000bp. Hence, what criteria can I rely on while testing the specificity of my primer sequences?
I sincerely appreciate your valuable contribution to answering my question.
I think your strategy is ok, but as you are using qRT-PCR, for the Primer Blast select a database corresponding to transcriptome (i think you are using genome, it shows you chromosome location, and maybe your primers are on exon-intron boundaries so you don't find your gene and the sizes will be different). GL
A few things:
Daniela Liebsch You're absolutely right! When I changed the database setting from "Refseq representative genome" to "Refseq RNA," it actually displayed the primers I wanted exactly as I had selected them from NCBI nucleotide.
Jakub Jagielski Actually, I didn't perform a PCR using a cDNA sample to validate the specificity of my primers. Instead, following your suggestion, I designed the primers using NCBI nucleotide for the specific sequence I was interested in. I focused on the mRNA and CDS regions and used another software to assist me in selecting the primers.
To theoretically verify the specificity of the primers, I utilized NCBI Primer Blast. I inputted my newly designed forward and reverse primers and requested the system to retrieve the primers. Subsequently, I compared various factors such as product size, gene name, Tm, and other parameters with my own designed primers.
As for the supported Ezrin sequence, it's possible that the associated non-specific product size, "desmocolin2," is considerably larger (around 1400) compared to the length of the Ezrin product (approximately 300). This disparity might suggest that a longer duration of PCR is needed for the production of the "desmocolin2" product, which could potentially explain how they managed to obtain the primer product of their interest. Perhaps someone could provide a reasonable explanation for this issue.