3 replies to this topic

[Bill Nye]

Hi,

I am doing a luciferase reporter assay. I would like to have a control for transfection efficiency, and it seems like co-transfection with a reporter plasmid is the way to go. I was thinking of transfecting the luciferase plasmid with a selectable vector (both plasmids being relatively the same size ~5.5kb).

A couple of questions:

1) I am assuming that, via selection
...a) both plasmids enter the cell
... only one plasmid enters the cell

situation a) would be ideal
situation would suck if only selection vector entered the cell, but luciferase did not

Co-transfections are never perfect, but in your opinion, is it good enough? If not, what else would you suggest?

[avitas]

some people would cotransfect with a constitutively active renilla reporter as control if you are using firefly luciferase reporter for your study. however, you would be assuming the cotransfection efficiency is similar for both reporters.

[Rsm]

Yes, the usual way to go is the dual luciferase assay, at least as far as I know... I have never done this and will never do, because there are much better ways to do this measurement rather than Luciferase. This technology is just an old fashioned relict of the days where GFP was not available.

If you're using GFP and a flow cytometer (FACS Canto or whatever) you can accurately check if a single cells expresses your reporter,
expresses your gene of interest (GOI) and also if there is a dose-dependency. And you can do this for 10000 cells per second. However, not everyone has access to a flow cytometer...

In my experience, co-transfection with a reporter does not provide a measurement of transfection efficiency of your GOI. I reproducibly found single positive cells at around 25% of all transfected cells (again, measured with a FACS). So I would not recommend Dual Luciferase, simply because it's noisy. But it's approved, and you won't have to fight with your supervisor if you want to do it.


Cheers,

Minna

[paulis]

Hi everyone,

I'm doing a Renilla luciferase assay to measure the activity of protein promoter "A", and I'm transfecting with another plasmid, which is the protein "A" fused to GFP, so my question is, if I have some measures problems when measuring luciferase activity with luminometer, I mean GFP could interfere with the measurement of luciferase?

Please help me