Northern Normalization

Ned Mantei mantei at neuro.biol.ethz.ch
Thu Aug 22 01:00:05 EST 1996


In article <321B12ED.304E at rzmail.uni-erlangen.de>, Heinz-Juergen Schaefers
<mfm434 at rzmail.uni-erlangen.de> wrote:


> the best internal standard for RNA loading is 18s or 28s rRNA.
> 

Beyond a certain amount of nucleic acid on the filter, the limiting factor
in hybridization is diffusion of the probe to the filter. Whether the
amount of rRNA on the filter is "100%" or only 20% of that amount might
not make any difference in the signal obtained. Therefore rRNA might *not*
be a good internal control.
My own choice is to use S1-mapping or RNase protection assays. You can
calibrate absolute amounts against a series of external standards (in
vitro transcript corresponding to the one you are measuring, covering at
least the region protected by the probe; isolate full-length transcript by
agarose gel electrophoresis and quantitate by UV spectroscopy). Include in
every RNA sample, natural and external standard RNA, a small amount of in
vitro transcript of a deleted template that gives a shorter protected
fragment--this controls for loss of sample during workup and loading the
gel. (For this transcript, those not-full-length cDNA clones will finally
be good for something!). Quantitation is especially easy with a
PhosphoImager or similar machine. You can then calculate with reasonable
accuracy how many pmols or ugrams of transcript you have per ugram of
total RNA.

-- 
Ned Mantei
Dept. of Neurobiology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
mantei at neuro.biol.ethz.ch   Fax: +41-1-633-1046



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