How to optimize microRNA specific primer?

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Frederic Lepretre

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

Shahrooz Ghaderi

Many Thanks Dear Frederic

Can Kiessling

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.

Rolf Søkilde

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.

Dear Frederic.

Thank you for your nice protocol.

Thanks Mikael.

I just download your method today. But please honored me, for good concepts later. I am eager to know the difference between your job and Peter Busk.