I`m getting 50-100 fold induction of my desired miRNA by inducible system. I was curious about the extent of reduction of luciferase activity.
Can you be more specific about the delivery systems you are using? There are different problems with different combinations. Here are some issues that most people in the filed tend to ignore...
If your Dox-miRNA is a stably integrated lentivirus and you are hoping to transfect a plasmid with a luciferase reporter, it won't work: transfection delivers multiple copies of plasmid into few cells, compared to the single integration of all cells in the lenti system. So in some of your cells there will be too much luciferase for the miRNA to suppress and in some of them there will be no luciferase at all. Your readout will depend on the transfection efficiency and not on biological miRNA activity.
If you are planning to co-transfect two plasmids, then luciferase will start expressing before the miRNAs had a chance to be transcribed and processed. Being a stable protein, this initial unregulated burst will severely mask your readout.
Your can induce the miRNA and transfect an in-vitro transcribed luciferase RNA reporter, but there are technical challenges. Or clone the reporter under a weak promoter and do a long term steady state measurement so that the initial burst of luciferase synthesis becomes insignificant. Or...
This is all assuming, of course, that endogenously processed pre-miRNAs act in the same way as mimics. Which they don't. :-)
All in all, a potentially complicated answer to a seemingly simple question!
Good luck,
A.
Can you be more specific about the delivery systems you are using? There are different problems with different combinations. Here are some issues that most people in the filed tend to ignore...
If your Dox-miRNA is a stably integrated lentivirus and you are hoping to transfect a plasmid with a luciferase reporter, it won't work: transfection delivers multiple copies of plasmid into few cells, compared to the single integration of all cells in the lenti system. So in some of your cells there will be too much luciferase for the miRNA to suppress and in some of them there will be no luciferase at all. Your readout will depend on the transfection efficiency and not on biological miRNA activity.
If you are planning to co-transfect two plasmids, then luciferase will start expressing before the miRNAs had a chance to be transcribed and processed. Being a stable protein, this initial unregulated burst will severely mask your readout.
Your can induce the miRNA and transfect an in-vitro transcribed luciferase RNA reporter, but there are technical challenges. Or clone the reporter under a weak promoter and do a long term steady state measurement so that the initial burst of luciferase synthesis becomes insignificant. Or...
This is all assuming, of course, that endogenously processed pre-miRNAs act in the same way as mimics. Which they don't. :-)
All in all, a potentially complicated answer to a seemingly simple question!
Good luck,
A.
Dear Anna, thank you very much for your reply and please excuse my delayed response. I have generated my miRNA (~500bp, ~200bp on both side) stable cell line through Clontech`s retroviral system (pRetroX-tight-pur+RetroX-tetOn-advanced vector). I can see the effect of miRNA on its target mRNA and protein. Then I tried to do the 3`-UTR reporter assay using that system. My UTR reporter construct is on a strong promoter (cmv-renilla) and I used pGL3 control vector for normalization. I cotransfect them using lipofectamine and after 4hrs I have changed the medium with doxycycline.
My results were same as you mentioned, transfection efficiency is not equal among replicates, some replicates show suppression of luciferase activity, some doesn`t.
Thank you again.
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