Quantitating Northerns on MD PhosphorImager

Jarkko Kortesmaa jkortes at paju.oulu.fi
Tue Jan 9 08:27:45 EST 1996


In article <D.J.Glover.54.0011509E at bham.ac.uk>, D.J.Glover at bham.ac.uk (David Glover) says:

>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?

I've had some concerns about the quantitation myself. It seems to me that 
I get results that fit best with "eyeball" quantitation WITHOUT backround
correction. If there is a strong band near a weaker band, it seems that
the strong band can be counted as backround of the weaker, sometimes 
resulting in negative intensities. I find it logical to think that 
the signal/backround ratio is sufficiently constant within the blot to 
allow comparison of, for example different tissues, even without any
backround correction. If the blot is pretty, that is.  

I have used selection objects of equal size for each band. Again, this
might not be as good an idea with "ugly" blots.
 
About comparing to actin, I would be very interested to know the idea 
behind it. Is there proof that the amount or proportion of actin mRNA 
is in any way constant? If not, what does probing with actin tell? Only
that the RNA was not totally degraded?

	
				- Jarkko Kortesmaa



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