How to "normalize"

Heinz-Juergen Schaefers mfm434 at rzmail.uni-erlangen.de
Wed Jul 31 14:37:07 EST 1996


lmrc. wrote:
> 
> When doing Northern studies, people use 28S or some constantly expressed
> house-keeping genes (such as actin in my case) as standards for loading and
> transferring.  How is this "normalizing" done?  If I would plot the
> expression of my gene-of-interest over time, how can I incorporated the
> expression of actin?  Is it simply by using time zero actin expression as
> one and actin gene expression over time as a fraction/ratio to time zero and
> then later use these values in multiplication?  Is there a simple
> corrolation between the standard and any gene-of-interest or is there some
> complicated formula I have to follow?  Any comments/warning is appreciated.
> Thanks!!
> 
> lank-mrc at tigger.jvnc.net

Hi!

To normalize you have to determine the OD ratio (suggesting u got the OD of the 
autoradiagraph bands by using a video desitometer or similar device) between the 
constant_expressed_gene and the gene_of_interest for each point separately!!!

Example:

Time course: 1,2,3,4,5 hours
for each time point you have the band OD for GOI (gene_of_interest) and CEG 
(constant_expressed_gene), so you have to get: GOI/CEG ratio for each of the 5 time points.
This ratio you now plot against the time...
Thats all...


BUT: Actin is not very good for normalisation due to some regulation upon several stimuli,
better use GAPDH (also sometimes regulated, but not so frequently as actin); the best is 
to normalize against 18s or 28s rRNA.

NOTE: to avoid crossreactions by simultanously probing with both GOI and CEG probes, or to 
avoid loss of signal by stripping the blot after first probing with GOI before reprobing 
with the CEG probe, you may cut the blot into 2 pieces (only possible if the probes run on 
different heights) and probe every piece with the appropiate probe...

bye
	Heinz
-- 
Dipl. Biol. Heinz-Juergen Schaefers
University of Erlangen, Med. IV, Nephrology Research Lab
E-mail: university: mfm434 at rzmail.uni-erlangen.de
           private: h-j.schaefers at erlangen.netsurf.de



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