Dear colleagues.
I recently designed some microRNA-specific primers. Can I use any software that optimized this method during in silico design? Because of the short length of microRNAs Tm optimizing getting harder at least for me. Does any body knows any specific software on this matter?
Hi Shahrooz,
miRNA are very small for designing primers, but you can try this site (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) with your own genome (your favorite miRNA...). or this site but you need to log-in (http://genomics.dote.hu:8080/mirnadesigntool/).
fred
You should first choose your cDNA synthesis kit.
There are different types of cDNA methods and your primer design will need to be compatible with that.
https://www.researchgate.net/post/What_is_the_best_technique_to_differentiate_pri-miRNA_from_mature_miRNA
Thanks Dear Can. You are right, However in microRNA-specific method we use traditional cDNA method including random hexamer and oligo dT. This is a cost effective method.
Hi Shahrooz,
I have used this method by Peter Busk and can highly recommend it.
A tool for design of primers for microRNA-specific quantitative RT-qPCR.
It is very like many commercial products, but you have more control of the design.
best,
Rolf
Dear Rolf.
Exactly!
I am using the Peter Busk Method. It is great. But in silico analysis of primers are different. However, the data from www.idtdna.com is closer to the Busk results than Oligo 7 and Primer Blast. Do you have any advise for me? Especially for Tm setting up?
Best Regards.
Shahrooz.Ghaderi.
Hi Shahrooz
a good PCR program that never failed for me is a touchdown PCR. chose e a range of 5°c before and after the optimum TM, begin at +5° and go down to -5°, -0.5° by cycle, 20 cycles, and add 20 more cycles at -5°.
this worked, this should work.
fred
Dear Frederic.
Thank you for your nice protocol.
Best Regards.
Shahrooz.Ghaderi.
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