We have been using OD measurements at 260 nm to estimate the copy numbers
of our RNA and DNA standards stock solutions for Q-PCR. However, we are
not satisfied by the precision and reproductibilities of the technique for
Q-PCR applications, that are requiring a very stringent estimation of copy
numbers. I am aware of some publications in Biotechniques relating to the
usefulness of 260/280 OD measurements to estimate RNA and DNA
concentrations, but OD measurements still seems to be, as far as I know,
the reference method.
Has someone encountered the same kind of problem; if it is the case, is
there a way to correct the OD readings (one way is suggested by Glasel in
Biotechniques18(1),62-63, but it seems to be adapted to the measurement of
DNA purity and not of DNA actual concentration), are there specific
precautions to take when performing the readings or is there a more
reliable method to measure nucleic acid concentrations than OD
measurements (Hoechst 33258 fluorescence would only solve our problem with
DNA, not with RNA). Has someone some useful readings to advice me?
Thank you for your help.