You need an internal control that you transfect at the same time as your
LUC (or CAT) test plasmid. This guy has to be put in the same amount
(often 5 ug) in every single plate of your experiment (except for your No
DNA negative control), so you'll want to make megapreps of it; just have
both plasmids (test and internal control) in the tube before you add
whatever you use to make them go in your cells
(e.g. HBS and CaCl2 for a CaPO4 transfection). Typically, this
internal control would be RSV-betaGal, but all you
really need is something that is expressed in your cells, that is not
modulated by whatever treatment you do to them, and that you can assay
conviently (and that is not the same as your reporter, of course).
An internal, co-transfected control is necessary because transfection
efficiency can vary quite a bit between plates. Thus, you express your
data as the ratio of the activity of the reporter gene driven by your
test promoter to that of the reporter expressed by your internal control
(often LUC/betaGal ratio). Try it and you'll see your duplicates merge
Following this correction, you may chose to express these as percentage
of a control LUC plasmid (itself corrected for betaGal too, of course).
Percent of a strong promoter (SV40, RSV, etc.) or fold increase of a
promoterless plasmid are both adequate. Though not necessary, this
sometimes helps comparison between experiments, if they are not well
reproducible, as may occur when you change a batch of plasmid, or of
luciferin/ATP mix, etc. With this respect, I prefer %RSV.
Hope this helps.
On 8 Oct 1996, Joe Miano wrote:
> Date: 8 Oct 1996 14:56:15 GMT
> From: Joe Miano <jmiano at post.its.mcw.edu>
> To: methods at net.bio.net> Subject: Best way to express Lucif activity?
>> I am sure this has been debated, but can someone comment on the best method
> of expressing luciferase (or for the matter CAT) data? I have been
> normalizing the raw RLUs to total protein and then express this value as
> either a percent of the SV40 control or as a fold increase over the basic
> (promoterless and enhancerless) vector.
> The Love you take is equal to the Love you make--P. McCartney
Dr. Louis H. Ferland
Centre de Recherche, Hotel-Dieu de Montreal
Dept de Nutrition, Universite de Montreal
Phone: (514) 843-2757 FAX: (514) 843-2719