Northern sensitivity

Helmut Dotzlaw dotzlaw at ccu.umanitoba.ca
Tue Mar 23 18:45:00 EST 1993


In article <01GVSY1CAWFK000E9B at irri.cgnet.com>, RSCOTT at IRRI.CGNET.COM
(RPSCOTT) wrote:
> 
> Blair Jennings at <HF.BXX at forsythe.stanford.edu>:
> >I'm trying to find out what is the lowest concentration of message
> >which can be seen on a Northern blot. Also how much more sensitive
> >is the RNase protection assay or is it? I want to know this because
> >I'm looking at a constitively expressed message during early
> >gestation and am having a hard time seeing anything other than my
> >positive control.
> 
> I don't know if you're using total RNA for gel running but it may be helpful
> if you load poly-A RNA instead. Now if the transcripts you wish to
> detect are indeed rare at the cell stage your looking, you may find it
> worthwhile to do reverse PCR. Try looking up the ff. paper:
> 
> S.A. Fuqua et al. (1990) A simple polymerase chain reaction method for
> detection and cloning of low abundance transcripts. BioTechniques 9(2):
> 206.
> 
> Hope I imparted something that will be useful for you Blair. ;-)
> Scott
> IRRI, Philippines
> e-mail:  rscott at irri.cgnet.com

I agree that selecting for poly A RNA improves the sensitivity in Northern
analyses, it's just kind of a pain to do and you need to start with quite a
lot of total RNA, I typically obtain about 4% poly A relative to starting
total RNA using oligo-dt cellulose.

We've been using RNAse protection assays to quantitate message in total RNA
samples, we find about a 10-fold increase in sensitivity over Northerns
using total RNA.  Initially we were using the protocol found in Current
Protocols in Molecular Biology, lately we've been using a kit (RPA II -
Ambion Inc.) - it works well in our hands and is less time-comsuming than
the CPIMB protocol)

If the message is extremely low abundance, then yes, RT-PCR, you need to be
careful though that it is "quantitative PCR", though - there is some
literature around, see Andre Hamel's posting in this SIG.

Hope you find something that works for you!

helmut

             Helmut Dotzlaw
Dept. of Biochemistry and Molecular Biology
         University of Manitoba
            Winnipeg, Canada
        dotzlaw at ccu.umanitoba.ca



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