Quantitative PCR

cindy boardman.3 at osu.edu
Fri Mar 17 11:28:30 EST 2000


Hello All!

Does anyone with experience using quantitative real-time RT-PCR have a
suggestion as to a good way to quantitate your standards?  My PI is
insistant upon being able to express our quantitative results in terms of
numbers of copies of message.  I currently use UV- spectophotometry and make
sure that my 260/280 is good.  But I am suspicous of trusting that i could
convert the OD 260 to -copies-.  Something about significant figures, and
having a blank which -exactly- represents the eluate.  I have resigned
myself to the idea that the -most- accurate way I can think of is to get
a -really- pure quantity of standard, and make serial dilutions, then
generate a curve for these.  Then calibrate my LightCycler standards to this
curve each time, as well as calibrate my Spec curve every time I want to do
another experiment.  The study is going to be conducted over several years
with clinical samples, so if we are to be able to compare this years data
with next years, we have to be certain that our standards don't drift,
....And we have to be able to detect on the level of -copies.

I am really in need of advise on the best way to do this.

Any suggestions would be greatly appreciated.

Cindy






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