Taek H. You
tyou at postbox.acs.ohio-state.edu
Sat Aug 24 00:38:34 EST 1996
In article <mantei-2208960800050001 at retrograde.ethz.ch> mantei at neuro.biol.ethz.ch (Ned Mantei) writes:
>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
The S1 mapping and RNase protection assays do not give the size information,
even though they are more sensitive assays than standard Northern blot.
The other way:
1. Duplicate lanes with same amount of RNAs (polyA+ or
total)2. Cut the filters in two
3. Probe each filter with two different probe, one of which is your specific
probe and the other is well known control gene.
I know some people used ribosomal protein gene probe in plants. Soybean actin
gene comes to mind also.
4. Using densitometer or computer analysis, you can normalize your signal
relative to the other control intensity.
Good luck on your research.
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