B-galactosidase variability in Luciferase assays - B-galactosidase variability in Luciferase assays (Jul/17/2008 )
Hi,
I'm having problems in b-galactosidase stability during luciferase assays. Typically we transfect cells (different cell lines) with Lipo2000 in 24wp using 0.15ug pGL3 luciferase-reporter gene; 0.1 ug SV40-bgal (or 0,025 ug pCMV-bgal) plus 0,3ug of our different repressors of interest (some of them also under pCMV control). We made equal the final amount of DNA/well with pBSKTT gene. We transfect them during 4-6-12h and change the medium and analyze by luminometryafter 48h .
We observed good luciferase signals (low background, 200-300.000 RLU).
We measure b-gal adding corresponding substrates (sometimes the half of the indicated reactive) and after 1h of incubation.
About b-gal... measures indicate high background signal (40.000) and low transfection (similar to background-although luciferase doesn't indicates this!) or good signals (300.000) in comparison to background but with great fluctuation (from 20.000-1x10^6). My boss thinks they should mantain constant.
So, what's happening? We are questioning everything! DNA quality seems to be good (A260/280=1.8) although differences in bacteria transformation have been seen depending on which plasmid you're growing. Is b-gal a good indicator of transfection efficiency or is it also modulated? Low backgrounds are typically solved doing 1h at 50ºC. However, when we did it, everything was "dead". We have also compared different kits (Clontech, Roche, Tropics) and although the signal changes the proportion is mantained. Why does b-gal change depending on the cotransfected plasmid? Looking at the bibliography some people offer alternative techniques such as Slot blot, PCR...which involve quite a lot of work for a periodical technique.
We are also thinking about using Renilla as measure of transfection efficiency; although some workmate has also observed variations in presence of TGF-b.
It's kind of a common technique, and any help would be really appreciated!!
Thanks a lot!
I'm having problems in b-galactosidase stability during luciferase assays. Typically we transfect cells (different cell lines) with Lipo2000 in 24wp using 0.15ug pGL3 luciferase-reporter gene; 0.1 ug SV40-bgal (or 0,025 ug pCMV-bgal) plus 0,3ug of our different repressors of interest (some of them also under pCMV control). We made equal the final amount of DNA/well with pBSKTT gene. We transfect them during 4-6-12h and change the medium and analyze by luminometryafter 48h .
We observed good luciferase signals (low background, 200-300.000 RLU).
We measure b-gal adding corresponding substrates (sometimes the half of the indicated reactive) and after 1h of incubation.
About b-gal... measures indicate high background signal (40.000) and low transfection (similar to background-although luciferase doesn't indicates this!) or good signals (300.000) in comparison to background but with great fluctuation (from 20.000-1x10^6). My boss thinks they should mantain constant.
So, what's happening? We are questioning everything! DNA quality seems to be good (A260/280=1.8) although differences in bacteria transformation have been seen depending on which plasmid you're growing. Is b-gal a good indicator of transfection efficiency or is it also modulated? Low backgrounds are typically solved doing 1h at 50ºC. However, when we did it, everything was "dead". We have also compared different kits (Clontech, Roche, Tropics) and although the signal changes the proportion is mantained. Why does b-gal change depending on the cotransfected plasmid? Looking at the bibliography some people offer alternative techniques such as Slot blot, PCR...which involve quite a lot of work for a periodical technique.
We are also thinking about using Renilla as measure of transfection efficiency; although some workmate has also observed variations in presence of TGF-b.
It's kind of a common technique, and any help would be really appreciated!!
Thanks a lot!
I have done it both ways and found renilla-luc to be more reliable, not sure of tgf-beta effect though.
I'm having problems in b-galactosidase stability during luciferase assays. Typically we transfect cells (different cell lines) with Lipo2000 in 24wp using 0.15ug pGL3 luciferase-reporter gene; 0.1 ug SV40-bgal (or 0,025 ug pCMV-bgal) plus 0,3ug of our different repressors of interest (some of them also under pCMV control). We made equal the final amount of DNA/well with pBSKTT gene. We transfect them during 4-6-12h and change the medium and analyze by luminometryafter 48h .
We observed good luciferase signals (low background, 200-300.000 RLU).
We measure b-gal adding corresponding substrates (sometimes the half of the indicated reactive) and after 1h of incubation.
About b-gal... measures indicate high background signal (40.000) and low transfection (similar to background-although luciferase doesn't indicates this!) or good signals (300.000) in comparison to background but with great fluctuation (from 20.000-1x10^6). My boss thinks they should mantain constant.
So, what's happening? We are questioning everything! DNA quality seems to be good (A260/280=1.8) although differences in bacteria transformation have been seen depending on which plasmid you're growing. Is b-gal a good indicator of transfection efficiency or is it also modulated? Low backgrounds are typically solved doing 1h at 50ºC. However, when we did it, everything was "dead". We have also compared different kits (Clontech, Roche, Tropics) and although the signal changes the proportion is mantained. Why does b-gal change depending on the cotransfected plasmid? Looking at the bibliography some people offer alternative techniques such as Slot blot, PCR...which involve quite a lot of work for a periodical technique.
We are also thinking about using Renilla as measure of transfection efficiency; although some workmate has also observed variations in presence of TGF-b.
It's kind of a common technique, and any help would be really appreciated!!
Thanks a lot!
did you mean 200-300,000 RLU or 200 to 300 RLU?
assuming you have 200,000 RLU, maybe you should use more beta-gal construct in the combination instead. Try 0.1 with cmv-b-gal, sv40-b-gal is much less active.
I have also observed variations in b-gal activity when cotransfecting a plasmid encoding p53. However, it has been shown a long time ago that p53 can repress viral promoters (such as CMV, i don't know about SV40 though), so you may have a similar situation in your setup. What you could do is change the promoter of the beta-gal...
I'm having problems in b-galactosidase stability during luciferase assays. Typically we transfect cells (different cell lines) with Lipo2000 in 24wp using 0.15ug pGL3 luciferase-reporter gene; 0.1 ug SV40-bgal (or 0,025 ug pCMV-bgal) plus 0,3ug of our different repressors of interest (some of them also under pCMV control). We made equal the final amount of DNA/well with pBSKTT gene. We transfect them during 4-6-12h and change the medium and analyze by luminometryafter 48h .
We observed good luciferase signals (low background, 200-300.000 RLU).
We measure b-gal adding corresponding substrates (sometimes the half of the indicated reactive) and after 1h of incubation.
About b-gal... measures indicate high background signal (40.000) and low transfection (similar to background-although luciferase doesn't indicates this!) or good signals (300.000) in comparison to background but with great fluctuation (from 20.000-1x10^6). My boss thinks they should mantain constant.
So, what's happening? We are questioning everything! DNA quality seems to be good (A260/280=1.8) although differences in bacteria transformation have been seen depending on which plasmid you're growing. Is b-gal a good indicator of transfection efficiency or is it also modulated? Low backgrounds are typically solved doing 1h at 50ºC. However, when we did it, everything was "dead". We have also compared different kits (Clontech, Roche, Tropics) and although the signal changes the proportion is mantained. Why does b-gal change depending on the cotransfected plasmid? Looking at the bibliography some people offer alternative techniques such as Slot blot, PCR...which involve quite a lot of work for a periodical technique.
We are also thinking about using Renilla as measure of transfection efficiency; although some workmate has also observed variations in presence of TGF-b.
It's kind of a common technique, and any help would be really appreciated!!
Thanks a lot!
Thanks for the help! Yes, it's probable that our transcription factors are modulating these promoters. We were thinking about RSV promoter.
Thanks!
I'm having problems in b-galactosidase stability during luciferase assays. Typically we transfect cells (different cell lines) with Lipo2000 in 24wp using 0.15ug pGL3 luciferase-reporter gene; 0.1 ug SV40-bgal (or 0,025 ug pCMV-bgal) plus 0,3ug of our different repressors of interest (some of them also under pCMV control). We made equal the final amount of DNA/well with pBSKTT gene. We transfect them during 4-6-12h and change the medium and analyze by luminometryafter 48h .
We observed good luciferase signals (low background, 200-300.000 RLU).
We measure b-gal adding corresponding substrates (sometimes the half of the indicated reactive) and after 1h of incubation.
About b-gal... measures indicate high background signal (40.000) and low transfection (similar to background-although luciferase doesn't indicates this!) or good signals (300.000) in comparison to background but with great fluctuation (from 20.000-1x10^6). My boss thinks they should mantain constant.
So, what's happening? We are questioning everything! DNA quality seems to be good (A260/280=1.8) although differences in bacteria transformation have been seen depending on which plasmid you're growing. Is b-gal a good indicator of transfection efficiency or is it also modulated? Low backgrounds are typically solved doing 1h at 50ºC. However, when we did it, everything was "dead". We have also compared different kits (Clontech, Roche, Tropics) and although the signal changes the proportion is mantained. Why does b-gal change depending on the cotransfected plasmid? Looking at the bibliography some people offer alternative techniques such as Slot blot, PCR...which involve quite a lot of work for a periodical technique.
We are also thinking about using Renilla as measure of transfection efficiency; although some workmate has also observed variations in presence of TGF-b.
It's kind of a common technique, and any help would be really appreciated!!
Thanks a lot!
did you mean 200-300,000 RLU or 200 to 300 RLU?
assuming you have 200,000 RLU, maybe you should use more beta-gal construct in the combination instead. Try 0.1 with cmv-b-gal, sv40-b-gal is much less active.
Hi,
yes it's 200,000-300,000 RLU. We will try with increase amounts of CMV-bgal; however, we are affraid to promote transcription factor competition or arrive to a plateu of detection. I will check detection limits in this luminometer.
Thanks for you help!
the use of cmv-b-gal is to normalize transfection variation between wells. it usually doesnt interfere that much of a specific transcription factor. The goal is that you need to see enough enzymatic activity to differentiate transfected vs untransfected background level. If you are getting high readings for b-gal, tune it down.
Thanks for your idea! Today I read something about in Gene Therapy 1997, 4:1313 - where they cited that " transfection causes cells to transiently arrest in G1 and induces p53" from Oncogene 1995, 10:1875 in murine fibroblasts doing calcium phosphate transfection. So, I don't know about lipofectamine but it reminds me what you observed!
Thanks