Question for dual luciferace reporter assay - -need a reporter for transfection efficiency (Oct/12/2005 )
I have some problems in my study and I need your help!
I used a dual luciferace reporter to evaluate tranlational regulation, cap-dependent and cap-independent. The signal of this reporter plasmid is not stable and sometimes the value is really low. It was not easy to collect enough data to do statistic analysis. I think I need another reporter to quantify the transfection effeciency of each assay.
Is there any suggestion? How about ß-galactosidase?
Thanks in advance
i'm really not sure, but does the Bgal functions in mammalian cells?
i would advice you to use DsRed protein. It's normally less toxic as GFP as a colleague told me. And she recommend me this one for Facs sorting...
Just an idea...
Clontech offers a lot of fluorescent proteins that can be used to detect a lot of stuff, the excitation spectrum of some proteins doesn't overlap with others (same goes for emmision spectra), so they are convenient for double labbeling purposes. Only problem is that you need several lasers in your FACS (or you can try fluorometry, but that one isn't really as sensitive in our experience). Be carefull also in your choice of reporters, most of them aren't monomeric (which can be a problem for fusion constructs and so on). I myself have some good experience with EGFP and DsRED2, and am going to try HcRED1 (because of excitation spectra of EGFP and HcRED1 are almost completely non-overlapping).
Bgal does function in mammalian cells as far as I know.
Isn't it possible btw to just use 1 luciferase construct and perform your experiment twice? One time cap-dependent and the other time independent?
Thanks for the suggestion
One more question!
Is the reading of the fluorescence protein, DsRed or GFP, could be quantify? or is there other plasmid detected by colometric analysis suitble for my assay?
I used to have an internal control in my assay to calibrate the difference between samples.
In this assay, the data was calculated as the reading of firefly/rhenilla. The reading of this plasmid is relatively low. I think this assay should use an internal control although I did not find it in my reference paper.
I am not sure whether it is correct......welcome discussion
which renilla construct are you using? Promega makes three different construct, SV40, CMV and TK promoters. I have found that the CMV is the strongest followed by SV40 and TK is the weakest. Once you add your lysate to the renilla substrate the signal generated isn't very stable so you just have to work quickly or use a luminometer equipped with injectors. Hope this helps!
i think you want just to know if your cell is transfected. moreover, expression of DsRed can change in two different cells even if they are transfeced in same conditions...