Using collage to analyze blot

Monica Magidin monica at lya.fciencias.unam.mx
Sun Apr 28 13:19:27 EST 1996


I am a biology student currently doing some work with Northern Blots
in order to analize the expression of specific genes but I've been
having a lot of problems with the mRNA loading.  I know that Ethidium
Bromide fluorescence is not a linear function of RNA (or DNA)
quantity,however I am not sure how this works for the emissions of the
radioactively marked probes (P32, in CTP). In short, I would be really
grateful if anyone would be able to answer me one (or all) of the
following questions:

- How can I calculate the exact amount of RNA I have to load into a
gel in order for it to be the same for all the samples, based on the
ethidium bromide fluorescence and O.D. of the sample, since these are
not linear functions (I know it sounds simple but I have been having
LOTS of problems).

-How can I analize the data I have if there was not an equal loading
of the samples?. I have been using the Collage software, and got up to
the point where I can calculate pixel intensity for the different
areas.  However I am not sure if there is a "correct" way to normalize
each blot against it=B4s own control.

       Thanking you in advance,
                Monica M.





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