What we pretend are primers for qRT-PCR.
The secondary structure of ncRNAs makes it difficult to design primers with regular programs such as Beacon ins't it?
Have you tried primer 3? It may be useful as it lists a number of considerations including secondary structure considerations for the primer itself, its worth a try putting in your ncRNA. I have never tried using it for this purpose though.
Primer blast should also work well. The cDNA will have less secondary structure than the RNA. Make sure you perform RT with random hexamers if your RNA doesn't have a poly(a) tail though.
Thank you very much. I will try out both ideas and evaluate which one can be the best in terms of design.
Tha random hexamers tip will spare us a lot of work. Thank you
I am not familiar with any specific programmes for ncRNA primer design, but if the secondary structure of RNA and/or amplicon is your concern, you can try any secondary structure prediction online tools. I found UNAfold from IDT (http://eu.idtdna.com/UNAFold?) quite useful. Just paste your sequence, set the temperature (normally 60C for qPCR) and see if your sequence forms stable secondary structures at this temperature. You generally want your dG value to be not too negative (and no more negative than -9 kcal/mol). Hope this helps!
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