I am trying to clone mature micro RNA-214 sequence in pcDNA3.1(-) and want to over express in NRK52E rat cell line.Before over expressing i want to check endogenous level of ''mature'' micro rna and after over expression also want to check level of ''mature" micro rna.Can anyone suggest me reliable kit for mature micro rna detection? For real time PCR can anyone suggest me how should i design primers for mature miR-214?
Firstly, you need to clone the pre-miR and not the mature miR. You need to allow the pre-miR to be expressed and to be processed properly by Dicer into the mature form. Secondly, I recommend you use the pCMV-MIR Vector for this cloning and not pcDNA, as it works much better for this kind of expression. And finally, to check endogenous levels of your microRNA, you should use Taqman qPCR - all the primers and probes are pre-designed for all the known miRs in the genome already. Good luck!
hi, you can check the expression of mature miRNA by two methods, stem loop and poly A polymerase, for the last one for example exiqon www.exiqon.com/microrna-real-time-pcr is the bast kit and you dont need to design the primers; but for the first one you should design your primers by yourself and its very easy there are many information about its protocol in papers.
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