I am trying to optimize a primers extension assay with 2-3 kb RNA fragments to study RNA modifications (methylation and pseudouridylation).
I was wondering if anyone has an idea about the minimum length of primers that can be used without compromising the annealing and the specificity.
Secondly is there a minimum distance that should be kept from the annealing site to the site to be analyzed.( this could also be considered as a minimal length that the RT enzyme would extend from the site of annealing)
Are you preparing cDNA first, and then a PCR or do you do one step RT-PCR?.
I work with viruses and regardless the kind of RNA virus I work with, random primers to do cDNA work well. You can also use a reverse primer from a specific set of primers to do cDNA and test at diferent temperatures from 37-42°C to see which temperature works better with that reverse primer (obviously it depends also on the kit or enzyme that you use to do cDNA).
I can say that a minimun length for primers is 14-16bp, but for sure the °Tm for a primers with that size won't be that high to make sure that you will get only specific products and PCR optimization may be required. However, you could increase the °Tm of that primers adding 5' non complementary extensions in order to increase the final °Tm of your set of primers.
Thanks Alejandro, i am doing a one step RT.
a 14 base primer sounds great and should do fine in my case as i do not use whole cell RNA for my analysis. A purified RNA is used as a template and as you said little optimization should do the trick.
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