dual glo luciferase assay+ pRLTK transfection problem - (Oct/25/2006 )

Hi everybody

I am performing luciferase assay by using dual glo kit from Promega. 293T experiments were great.( High readings for both plasmid) But when I start to use reasonably difficult to transfect cells ( like M17 and SHSY5Y) I start to get much lower renilla readings.( lucifease readings are still ok) So low that sometimes only 2X of background. Anybody have experience? My kit, plasmids etc. are all fresh.

The only explanation I might think of is that 293T cells has SV40 antigen and PGL3 plasmids have SV40 based sequences in contrast to pRLTK plasmid having HSV promoter. Does it make sense?

thanks in advance

Burcu

the reason is the SHSY5Y cells. These cells r difficult to transfect as u pointed out so u deliver few plasmids compared to the 293Ts where u deliver much more plasmid. And more DNA and more cells means more luciferase is produced and higher values.

I have found the same problem with my cells that have a 14%transfection efficency (sp?). The renilla will decrease as the amount of other DNA (ie luciferase containing DNA) increases. Also, pRL-TK is the weakest renilla expression vector from Promega. They have an SV40 and a CMV renilla, but I found that they interfere with the induction of my luciferase construct because they are so powerful and my promoter driving luciferase is relatively weak. Promega does say in the info sheets for their constructs that the promoters from each of the DNAs transfected into a cell can interact with each other and either enhance or mask activity of eachother. Good luck!