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RNAse protection assay?

Helmut Dotzlaw dotzlaw at ccu.umanitoba.ca
Mon Aug 2 12:49:12 EST 1993


In article <1993Jul22.185527.1260 at nphi.fi>, tronni at nphi.fi wrote:

> Hello all bio-netters!
> 

> 
> I'm considering the "RNAse protection assay", where you
> hybridize 32P-labeled antisense-RNA probes with target
> mRNAs in solution. I've read from many sources that 
> it should be more sensitive than Northern.
>

RPA is indeed much more sensitive than Northern, at least ten-fold in my
experience.
 
> Recently I saw in Biotechniques an advertisement by Ambion about their
> kit, which allows the reaction to take place in a single
> tube and you can even use multiple probes to detect many mRNAs
> simultaneously from total RNA.
> 
> I would be grateful for any hints and opinions about
> this. Should I get a kit or build my own?
> Is this assay really as good as advertised? Do the signals
> get too hot to be comfortable to work with?
> 
I am in no way affiliated with Ambion, but after years (thousands) of RPA's
using my own reagents, with inconsistent results and the like, I would
strongly recommend that you try the Ambion RPA II kit.  The one big
advantage over the conventional protocol is that there is no phenol
extraction involved to remove the Proteinase K (messy and lossy).  I have
had consistent and reproduceable results using this kit, as measured by
standard curves of in-vitro synthesized RNA. 

You will of course need a probe in a transcription vector, the probe should
be between 150 and 300 bp in length (hybridization efficiency decreases
dramatically with increasing probe length).  I would suggest that you
linearize the construct using an enzyme leaving a 5'-overhang, I have had
bad luck with 3'- and blunt ends, undigested probe in the sample lanes.  I
prefer to use T7 RNA Pol, just a personal preference, I have used SP6 and
T3 as well, it just seems that T7 is more consistent.  I have also found
that the supplier of labelled nucleotide (I use CTP mostly) plays a role -
I have had bad experiences with a particular supplier, the transcription
would work once, but once the label was thawed, that was it - no more on
the second try. Label from a different supplier works consistently through
freeze/thaw cycles. I won't post names, if interested, e-mail me. 

The signals will not be too hot to work with, typically I use 10 ul of
label to make the probe, this is usually enough to assay around 100
samples, so actually less label involved than a couple of standard
nick-translations.

Hope some of this helps, if you need more don't be a stranger!

Regards,

Helmut



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