RNase protection assay and radiolysis

Pamela Norton pamela.norton at mail.tju.edu
Wed Jan 2 15:28:53 EST 2002


In article <5.1.0.14.2.20011226115626.009ef200 at mail.albany.edu>, James
A. Dutko <jd4300 at albany.edu> wrote:

> Thanks in advance for any insight and advice.
> 
> I am performing RNase protection assay with gel purified in vitro 
> synthesized transcripts.  I label the riboprobes with UTP to specific 
> activities of 1-2 X 10e8 per microgram - the message is rare.  One probe is 
> 500 nt (protects 465 nt) and the other is 350 nt (protects 258 nt) long 
> each with approx 25% U's.  I store the probe in 0.5M NH4OAc, 0.2% SDS, and 
> 1mM EDTA at -80C.  After about 3 or 4 days the probe no longer detects the 
> messages.  Could this be due primarily to radiolysis of the probe?  Could I 
> extend the life of my probes by changing the storage conditions?  If 
> radiolysis is the nature of the beast for riboprobes then I can set up the 
> experiments differently.  Thanks again.
> 
> James
> 
> ---

I think that radiolysis likely is significant. What fraction of the U's
do you expect will be labelled? 

You can determine the amount of intact probe by running a small amount
on a gel after 3-4 days - how does it look? If you really need probes
of high specific activity, you might need to re-synthesize for each
experiment, sorry.

Pam Norton




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