CAT assays-reproducability

Michael Holloway ca566 at cleveland.Freenet.Edu
Fri May 28 18:32:08 EST 1993


>     I am engaged on a project in which I am attempting to analyse various
>putative enhancer elements for response to oncogenes (eg src, int, hst etc).
>We are currently using a reporter system based on the HSVtk promoter ie 
>pBLCAT2. The big problem has been reproducability of the response. I seem to be
>able to get everthing from 30-fold induction down to nothing from experiment
>to experiment. Is this level of variation unusual? If so, any ideas on
>minimising it? 
>  Another problem is that the negative control ie pBLCAT2 containing no added
>enhancer, appears to respond up to 10x to oncogene transactivation, minimising
>any putative enhancer effects seen on the elements under test. 
>  Several other groups use this reporter system with no such apparent problems.
>  We have tried controlling many perceived sources of variation with little
>lasting success, I can go into more detail with any respondents.

`Fraid I'm going to have to be tiresome and ask YOU for detail.  What
method of DNA introduction are you using?  It makes a big difference if
you're using lipofectin, calcium or electroporation.  Lipofectin is
supposed to be the most reproducible.  I know myself that 3 to 4 fold
variation with electroporation is not uncommon.  Calcium phosphate can go
sour very quickly if your reagents are compromised.  

You say that you're using a reporter with a promoter for a negative
control.  Why?  If you want something that will just show background
without any response you need something like pCATbasic or pSV0CAT.  If
you're just comparing the response to one enhancer to one oncogene to the
response of the same plasmid without the enhancer then what's the problem?

E-mail: mhollowa at (mail to freenet is forwarded)
phone:  (516)444-3612

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