Quantitating Northerns on MD PhosphorImager

David Glover D.J.Glover at bham.ac.uk
Fri Jan 5 17:18:46 EST 1996


Dear PhosphorImager Experts,

I'm about to start quantitating lots of bands on lots of Northern blots 
using a Molecular Dynamics PhosphorImager. I'm more familiar with ImageQuaNT 
but could also use IQ 3.3. I'd like to make sure I go about it the right way 
from the start, so any advice would be much appreciated.

I'm interested in quantifying samples for comparison with other samples on the 
same blot (all to be referenced to actin ultimately). The samples differ 
greatly in mRNA quantity and degrees of degradation (some have quite long 
tails). Here are my questions:

Should my selection object include these tails or just surround the 
main body of the band? 

Should the selection object be the same size for all samples on the same blot 
or, as the MD manual suggests, tightly surround bands whether broad or narrow?

What's the best background setting to use. I've just rooted out an old posting 
that says the "sum above background" in the old IQ 3.3 is bast. True?

Do these things really matter?

I'm not just being lazy and have tried all sorts of different combinations. 
They do give different results so any thoughts greatly appreciated.

Thanks in advance,
David Glover



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