naked shRNA transfection - (Nov/07/2008 )
I hope someone will help me with my problem.
I want to introduce to HeLa cells unmodified RNA resembling pre-miRNA. But doing a Northern Blot on transfected cells I can see only a degradation, and not specific cleavages e.g. by Dicer.
Does anyone have experience in transfecting cells with naked RNA? Should it be so easily degradeted?
What amount should I introduce, not to saturate the miRNA biogenesis machinery?
Waitng for any suggestions.
If the pre-miR forms shRNA structure it should be pretty stable. We have actually synthesized ~70-nt pre-miR and transfected into human cancer cells using lipofectamine 2k and observed similar effect as transfecting mature miRNA. We transfected it at a concentration of ~50 nM, but did not check whether it's actually processed into mature miRNA. Some groups must have done such experiment a while ago. Search PubMed with "mirna processing dicer kim vn".
Thanks for suggestions!
I found one paper in which similar experiment was performed in McManus lab.
But it is difficult to distinguish between Dicer products and degradation of RNA.