Hi Hiral

the best way to design qPCR primers is to get the mRNA sequence and mark the exon boundaries just to be sure to approximately share on of the primers on 2 exons. Amplicon length must be around 100-150Bp, not more. Tm difference between primers must be under 2°c and it's better to get the most GC content at 5' and 3' and avoid interactions between primers.

please use the UCSC website (http://genome.ucsc.edu) to get the most data as you can.

for my part I use Oligo7 to design primers, a licensed software, but it works fine and get more than 99% reliability.

all the best

fred

We can select this type of primer from RNA sequence because used in synthesis of cDNA

You only need one primer to span the exon-exon boundary to exclude gDNA. I'd suggest using the cDNA sequence as your input for primer design, trim it down to just the region you really care about, and see if what you get. Basically, give minimal sequence to force a design in that region.

Or, you can manually pick a sequence that spans an exon-exon-junction, use that as a known in Primer3 and look for a second. More of a "brute force" approach.

Good luck!

Thank you all for your suggestion. Even I am also selecting cDNA/mRNA sequence in IDT primer tool and primer 3 for designing primers , I am not getting perfect primer as i got unusual product when i use the same primer in UCSC in silico pcr to validate the primers. I don't understand what is happening.

maybe you need to select the right genome ?

fred

You can use Allele Id software . First retrieve the sequence information in genbank format from NCBI. Then you can design primer , through exox exon Junction method of the software .

You can use primer express software for qpcr primer designing. You may check you primer binding site through primer blast.

You might be detecting splice variants, if they exist between your primers. Just assume your product will be the size of the coding sequence MINUS the introns. Some intronic might get in there though. These guys can help with primer design: https://www.yourbiohelper.com/

Thank you all for your valuable suggestion. I have another query. When I design a primer from primer 3 and then validate my primers using primer blast it shows unintended templates other than intended ones. But when I design primers for the same transcript variant of a gene (with the help of pick primers from ncbi page) using primer blast, I got perfect results - only target template and no unindented products. Can any one explain me why this is happening as primer blast is a combined interface of primer 3 and primer blast. I am also attaching the screenshots of the same.

The best is to handpick the primers between the exon exon boundaries and match it to that of online tools like Primer3. That way, you would inherently not miss the regions you mask for. We always did that

-Prash