In order to see the functional activity of my miR-over expressing constructs, I did a co-transfection of the over-expressing plasmid with its target reporter. I could hardly see much difference between the mir-over+ mir-target co-transfection and empty vector+mir target co-transfection. I used a ratio of 1:1 for mir Over/empty vec to mir-targets. Does the miR-over plasmid has to be titrated for a better concentration and/or ratio?
Absolutely, the ratio should be tested in detail for an oprtimal result of miR-mediated target expression inhibition.
There are a few things outside of the plasmid ratio to consider.
What is the endogenous expression level of the miR in your cell line?
What is the reporter and what time point are you using for your assay?
What promoters are on your miR-expressing and reporter constructs? Are these strong or weak promoters in your cell line?
Finally, how are you determining your transfection efficiency? Is it fairly high? This may not be important for the current reporter assay, but will be important for your functional studies with miR overexpression.
Thanks to you both for the response!
@Andreas, any suggestion on an ideal ratio to start with, apart from 1:1?
@ Doris, a comparison of the sponges transfected alone in the Hela cells with the empty vector with no targets, gave me varying activity levels for each of the miRs. Some were expressed really high relative to the reporter vector with no targets in 3'UTR, some did not show any reduction in the reporter activity suggesting negligible or no expression in the cell line tested. BTW, how does the endogenous expression level matter for my over-mir plasmid to down-regulate the targets cloned reporter (luciferase)?
Reporter gene is luciferase expressed from PGK promoter while miRs are expressed from CMV, both seem to be quite strong in Hela. The time point I used is 24 hrs post transfection ( I had imaged the luc activity at 48 hrs time point too, but that was not different from the 24 hrs tp).
Transfection efficiency...hm.. I used 0.5 ug PEI to 1 ug DNA and a simple FACS with GFP marker gave me an efficiency around 70%.
I would really appreciate any suggestion!
Thanks!
Sabna
Yes, I usually start with a ratio of 1: 2.3 or 1:2.5 (RNA/DNA : Lipofectamine(R)2000)
Thanks Andreas! But I was intending at mir target construct to mir over-expressing construct ratio. RNA/DNA to lipofectamine, I use 2:1.
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