Joe Miano wrote:
>> Thanks for the note. I have always wondered with suspicion about
> contransfections. Does this not introduce squelching and perhaps an
> under-representation of the true promoter activity? I have seen some labs
> not use an internal control plasmid but I think you're right if you say a
> majority of labs do.
>> What I typically do is transfections in triplicate (6 well dishes) and I do
> this at least 3 times. I am finding no significant difference between
> experiments when I express the normalized RLUs (normailzed to protein) as a
> percent of SV40 or as a fold increase over pGL3basic (which bythe way gives
> high background in many cells!). I do not see any profound argument
> against not using an interbnal standard although I can imagine some hostile
> reviewers wanting one to do it the "correct way."
>> Is there really a correct way of expressing the data?
>> I would encourage more people to comment on this issue that I have debated
> for some time know. I really do not know the answer.
> The Love you take is equal to the Love you make--P. McCartney
Another way to perform your experiment when it is possible is to perform
your transfection and trypsinize your cells the next day. Then you split
them, so you have real duplicate and the transfection efficiency is not
a problem. Of course, this is not possible if you have to compare
transfections with different plasmids.
Florence Cabon. CNRS IFC1, Villejuif, France.cabon at infobiogen.fr