how to analyze transfection efficiency of miRNAs? - (Jul/30/2007 )
I have a doubt....
I wanted to know how one can analyze the transfection efficiency of pre-miRNA into the cells?? or even transfection efficiency of siRNAs??
You could get labeled siRNA/miRNA. I have just ordered the cy3-labeled negative control Pre-miR from Ambion. Haven't tried it yet.
I think the use of labeled siRNA is mainly for tracking % uptake which usually is high and should be different from the rate of transfection, because a mass of the siRNA taken up by cells may not be biologically active. It is therefore an overestimation for the efficiency. Biological assay for gene knockdown would be the real test.
Using a biological assay for knockdown efficacy can be a problem because you need to carefully control for toxicity. If you see decreased intensity on a blot, that might mean you knocked down the target and it might mean you made the cells sick.
With steric-blocking oligos we typically use splice-correction assays, which upregulate a reporter signal (usually GFP or luciferase) on successful delivery into the cytosol/nuclear compartment. I don't know how to set up an upregulation assay for miRNA, but that would be a preferable system.
A two-component system might work. For example, plasmid expressing a lac repressor with a 3' miRNA target and a beta-galactosidase gene: you put an miRNA into the cell that targets the repressor and the galactosidase activity increases. Not a trivial system to set up, but it would make a nice part of a paper.