is it possible to do real time PCR for detection of pre/pri miRNA in given tissue type using syber green ?
Yes it is. But the approach if different. You have to isolate miRNA, polyadenylate it and then use an universal primer and a specific primer.
Agilent has a kit that can help you with polyadenylation and 1st strand synthesis
https://www.agilent.com/cs/library/usermanuals/Public/600036.pdf
thank you for your kind response, i am agree with it , but if i have the sequence with me and i can design primer for it and will go for cDNA synthesis through random hexamer and go for amplification as for normal real time PCR. will it not work in this case?
Sorry but I can't confirm it. I've never done that way neither I have heard about it.
Take a look at this article. Maybe it can help you
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2663046/
i have this article with me and i trying my best to do it , but i dont think anything looks illogical in above-mentioned approach. but i am worried about secondary structures present in case of miRNA for this amplification purpose. i think peoples doing such thing will be more helpful but unfortunately i am not in contact with any of them.
thank you Chinmaya Sadangi
thank you for your kind reply but please elaborate it up to some extent so that i can relate it to a proper reference you are sugessting about
thank you mikael, it was very interesting to know about this recent method as per your earlier sugession. i am looking in to it, do you have personal experience with this method or any other method for miRNA amplification.
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