I am planning to use the Exicon's LNA miRNA expression PCR mix based on SYBR Green technology to asses a miRNA expression levels. To this kit the LNA primers from manufacturer are designed, but I thinking about to use my own set of primers (specyfic forward and uniwrsal revere).
Did anyone try to use mentioned combination before? or know a manuscript where it was described?
You could probably just design the primers with the same sequence as in the kit and most likely this would work. LNA is designed to increase specificity and binds "more tightly" with the template. Therefore you will get better results with LNA probes. BTW, Exiqon does not provide the sequences of their primers AFAIK. So you would need to design them yourself.
Another option is to design LNA primers, you can order them from IDT and others.
Now if you want to take a complete DIY approach you will also need to do a polyadenylation step before RT, because miRNAs are typically too short to be detect with PCR techniques.
Good luck!
Dear Tilen, thx for your answer.
I will design the primers for the reference miRNA and next I will compare amplification efficiency both normal and LNA primers, because I read that LNA primers have an increased specificity, but on the other hand I heard that the PCR efficiency is decreased (efficiency range between 80-85%).
About polyadanylation step- it is include in RT step.
I only curious why anyone did not use before self-designed primers with this PCR system, I cannot find any paper described it.
Regards
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