microRNA knockdown its target by western blot but can't inhibit the 3'UT - want a reliable protocol to do the 3'UTR luciferase assay (Jan/31/2011 )

Hi everyone,
I overexpressed my microRNA in the tumor cell line and did see 50-80% knockdown of its target proteins by western blot. So just like every publication, I tried to confirm the specificity with 3'UTR luciferase assay. For this purpose, 293T cells were seeded in 6-well plates. Each well was co-transfected with 1 ug of microrna expression plasmid or empty control plamsid and 10ng of 3'UTR-luciferase plasmid, which also already has another internal version of luciferase as the internal control for the transfection efficiency. So my questions include:
1. when microrna was overexpressed by the plamid (either transiently or stable), how long does it take to mature? considering it has to be transcribed into precursor, then processed to mature microrna, by then 3'UTR luciferase has been overexpressed (or not) ? is it too late to inhibit luciferase activity if co-transfection was used? So just transfect 293T or other cells with microRNA Vector for 24h before adding 3'UTR luciferase construct.
2. Is commerical synthetic microRNA molecules good at this trick? Since it doesn't need to be transcribed from the plasmid but just to be processed by Dicer or whatever to mature microRNA. So that's why we see most of the publication use this kind of magic molecules to do the 3'UTR luciferase assay?
3.What's the appropriate ratio if I still want to do this 3'UTR luciferase inhibition with plasmid based microRNA expression vector?
4. How long should I make the PLB lysate if I use the co-transfection protocol for the 3'UTR luciferase assay?

I also noticed the bad reproducibility of microRNA in terms of knockdown its targets. Even missile can miss its target, right?


Appreciate any suggestions.

Who did you buy your 3'UTR luciferase vector from?

biotechgirl on Tue Feb 1 00:40:45 2011 said:

The 3'UTR luciferase vector is pMIRGLO from Promega. This vector contains both firefly and renilla luciferase.

littlecell on Tue Feb 1 15:05:03 2011 said:

I had issues with their vector - I think it was too large for our purposes, and ended up using using ones from here "http://abmgood.com/miRNA(microRNA)/3'UTR-miRNA-Reporters.html" They work really well, and they wouldn't tell me specifically, but they must have had a good reason for splitting the two reporter genes out.