luciferase RNA levels

Lloyd Scot Bastian Ph.D. yacman at u.washington.edu
Thu Sep 16 10:09:49 EST 1999


I have previously published experience in detecting luciferase message in
transiently transfected cells. (Bastian, L.S. and Nordeen, S.K,  Molecular
Endocrinology 5:619-627 (1991).).  Although I did succeed, in generating
reproducible data that demonstrated that message levels were roughly
proportional to luciferase activity levels, it took me many tries to isolate
message from Mouse L fibroblasts.  I really don't think it was worth the
effort.  The problem is, I believe, that luciferase message is not very
stable.  Furthermore, the protein is not particularly stable either, although
some of the new vectors have modified the coding sequence to increase
stability.  If I were in your shoes I would reconsider your rationale.
YMMV.

dixie at terryfox.ubc.ca wrote:

> We are transfecting luciferase reporter gene constructs into Cos cells and
> assaying for luciferase activity 48 hours after transfection.  We get good
> levels of protein activity but are having great difficulty quantifying the
> amount of luciferase mRNA in the cells.  The RNA levels seem very low and
> are hard to detect on a Northern blot.  The luciferase vector we are using
> is pGL2 from Promega. Has anyone experienced a similar problem or does
> anyone have any suggestions as to why the RNA level appears to be so low?
> Thanks,
> Dixie Mager




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