What do *YOU* call background?

Gil kgilbert at cmp.hmc.psu.edu
Thu Jan 25 15:11:34 EST 1996


Greetings colleagues.

I have been using northern analysis for the last couple of years,
examining gene expression in lung.  Usually, I use a Betascope 603 blot
analyzer by Betagen to quantify cpms from bands of interest, but the
question also applies to densitometric analysis of autoradiographs. The
question that has concerned me from day one is "what do you use as
background?"  Should one take a background sample from each lane, from
between lanes, above bands, below bands, or what?  I have been taking
"blanks" from 4-6 areas on the blot (uniform size sample box) and
averaging.  Then, I take the average "background" and subtract this
average from the bands of interest.  Sometimes, this background average
can be as high as a specific band, even though there is an obvious band
there.  Usually, some lanes have more background than others, so it
seems that each lane should be treated differently with respect to
background.  

Also, when bands of interest are normalized against a housekeeping
gene, the amount of cpms subtracted as background can have a huge
influence on the supposed XX-fold increase or decrease in gene
expression of that particular gene product.

I've yet to find any consensus opinion on the question of background at
this institution, and I'm wondering if there is any among your labs and
institutions.  As with many molecular biology techniques, when you ask
5 people the same question concerning methodology, you get 5 different
answers!  Your comments will be greatly appreciated.

Gil
Dept. Cell and Mol Physiol
Penn State Univ, Col of Med
e-mail: kgilbert at cmp.hmc.psu.edu



More information about the Methods mailing list