Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Luciferase transfection controls


  • Please log in to reply
3 replies to this topic

#1 Bill Nye

Bill Nye

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 24 March 2010 - 05:48 PM

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
...:wacko: only one plasmid enters the cell

situation a) would be ideal
situation :P 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?

#2 avitas

avitas

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 06 May 2010 - 09:14 PM

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.

#3 Rsm

Rsm

    Post Dog

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 381 posts
8
Neutral

Posted 07 May 2010 - 12:35 AM

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
I got soul, but I'm not a soldier

#4 paulis

paulis

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 21 November 2012 - 07:20 AM

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 Posted Image




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.