We work with the dual luciferase assay from Promega, using Renilla lucifearse activity as the transfection control. I wondered whether it would be better to have this driven by a weaker (TK) promoter, rather than by the stronger SV40 or CMV promoter? The aim is to use this in transient transfections in colon cancer cell lines, and I do not expect very strong induction of the firefly luciferase due to likely weak activation of my transcription factor.
Hi Berlinda,
My name is Robert Deyes and I work with Promega's Technical Support group. I agree with most of the points above although I generally recommend weaker promoters for a Renilla internal control. The reason is that you only need to be above background in order to normalize the firefly luciferase. 'Trans' effects such as the ones that quirin mentions in his reply, are more prevalent when strong promoters such as CMV are used for the Renilla Internal Control. so I would say the Tk promoter is a very good option (used at a 10:1 transfection ratio firefly:renilla). The Promega pGL4.74 plasmid will do the trick:
http://www.promega.com/resources/protocols/product-information-sheets/a/pgl474-vector-protocol/
Another interesting observation that many researchers have noted is that you can even get by without any promoter at all (ie you can still get cryptic transcription of the Renilla gene with a promoterless Renilla vector such as pGL4.70):
http://www.promega.com/~/media/Files/Resources/Protocols/Product%20Information%20Sheets/A/pGL470%20Vector.pdf
This might work better in cases where you are doing compound screens that elicit high levels of transcriptional activation (you do not want activation of the Renilla since that nullifies the whole purpose of using it as an internal control; so you remove the promoter to minimize the chances of that happening).
In summary, try the pGL4.74 first. If you do not get a robust Renilla signal, try pGL4.70.
Best regards,
Robert
Robert Deyes
Technical Services Scientist
Promega Corporation
Tel 1 800 356 9526 Ext 1366 (USA)
Tel 1 608 274 4330 Ext 1366 (Long Distance, Toll Paying)
http://www.promega.com
Email: [email protected]
We always use TK in our experiments and analyze the samples with Promega kit.
Good luck!
Hi Berlinda,
Actually I think your strong control renilla construct could also be advantageous. It allows you to minimize the amount to transfection control plasmid enabling you to use more of the promoter construct you are interested in.
cheers
Michael
Hi Berlinda,
My name is Robert Deyes and I work with Promega's Technical Support group. I agree with most of the points above although I generally recommend weaker promoters for a Renilla internal control. The reason is that you only need to be above background in order to normalize the firefly luciferase. 'Trans' effects such as the ones that quirin mentions in his reply, are more prevalent when strong promoters such as CMV are used for the Renilla Internal Control. so I would say the Tk promoter is a very good option (used at a 10:1 transfection ratio firefly:renilla). The Promega pGL4.74 plasmid will do the trick:
http://www.promega.com/resources/protocols/product-information-sheets/a/pgl474-vector-protocol/
Another interesting observation that many researchers have noted is that you can even get by without any promoter at all (ie you can still get cryptic transcription of the Renilla gene with a promoterless Renilla vector such as pGL4.70):
http://www.promega.com/~/media/Files/Resources/Protocols/Product%20Information%20Sheets/A/pGL470%20Vector.pdf
This might work better in cases where you are doing compound screens that elicit high levels of transcriptional activation (you do not want activation of the Renilla since that nullifies the whole purpose of using it as an internal control; so you remove the promoter to minimize the chances of that happening).
In summary, try the pGL4.74 first. If you do not get a robust Renilla signal, try pGL4.70.
Best regards,
Robert
Robert Deyes
Technical Services Scientist
Promega Corporation
Tel 1 800 356 9526 Ext 1366 (USA)
Tel 1 608 274 4330 Ext 1366 (Long Distance, Toll Paying)
http://www.promega.com
Email: [email protected]
- Badges
- Science topic