Sequencing small, non-coding RNA

Johns053 at mc.duke.edu Johns053 at mc.duke.edu
Sat Mar 17 10:52:47 EST 2001


You could go back and use the ancient procedure of enzymatic sequencing of RNA.
All you would have to do is 5' and 3' end label your RNA and then digest with
the panel of sequence specific RNases.  There was even a commercial supplier of
a kit to do this at one time (I don't remember whom).  If you check through the
Methods in Enzymology index, you can probably find a detailed protocol.  Even
though this is old-fashioned, it may prove easier than RACE.

Good luck,
Stewart




rjklein at genetics.wustl.edu (Robbie Klein) on 03/16/2001 10:02:26 PM

Please respond to rjklein at genetics.wustl.edu

To:   methods at hgmp.mrc.ac.uk
cc:
Subject:  Sequencing small, non-coding RNA



I'm currently studying several novel small, non-coding RNAs (approx. 125nt
in length).  I know the genomic sequence they are transcribed from, but
want to verify the sequence of the RNA to make sure there are no
post-transcriptional modifications.  Ideally, I'd like to do 5'- and
3'-RACE on the molecule.  As it's such a short molecule, the two fragments
would overlap and I could sequence each fragment to get the full sequence
of the RNA.  I've looked into the various RACE kits that are on the
market, and they all seem to be designed for eukaryotic mRNAs.
Specifically, the 5'-RACE kits have a separation step where the first
strand cDNA is separated from the oligo using a column before tailing.
However, the columns only work for cDNAs > 300nt long, which is impossible
when the total RNA length is 125nt.  The 3' kits assume a poly-A tail,
which my RNAs don't have.  I was wondering if anyone has a protocol for
PCRing up the ends of a small RNA without a poly-A tail.  Any other advice
you may have would also be appreciated.  Thanks.

-- Robbie







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