Hello Amira,
usually for qPCR or RT-qPCR you want short amplicons between 70 and 200bp (150 bp is considered ideal). You also want your primer to be average in GC content and to have an hybridation temperature close to 60°C.
If your are working on an eukaryote organism, RT-qPCR primer should also span an intron or an exon-exon junction to be sure to amplify your target retrotranscribed mRNA and not the DNA it came from.
If your PCR primer don't fit those criteria, you should design and order new ones.
After receiving them, do a qPCR to check for specific amplification (running melt curves on your qPCR apparatus, and at the end run your qPCR product on a 3% agarose gel). Check also for the efficiency of your primers by doing a qPCR on serial dilution of template.
here is an online tool to design qPCR primers:
https://eu.idtdna.com/scitools/Applications/RealTimePCR/default.aspx
I can't thank you enough for your information, Dr. Paget-Bailly.
actually, I'm not working on eukaryotes but viruses specially fish viruses. will the criteria of PCR primer differ from that of eukaryotes? I want to quantify the virus only to be used in an experimental challenge. Philippe Paget-Bailly
Amira Ezzat no problem !
So you will work on the virus DNA (assuming it is a DNA virus i don't know) ?
not on its RNA.
Basically, the thing I said about exon and intron is a precautious step to be sure that you don't amplify DNA when you target RNA. But there is a lot of case when it is not possible.
Yeah the same broad parameters apply for every qPCR basically.
The thing with viruses is that they can have genome enriched in AT or GC which may decrease or increase, respectively, the Tm of your primers. But in most cases the link I gave will give you the best possible combination of primer given your sequence no worries !
Philippe Paget-Bailly thank you for your prompt reply and information you provided, Dr.Paget-Bailly. It will be the first time to design a primer, so i can't thank you enough at all.
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