Sequencing small, non-coding RNA

Robbie Klein rjklein at genetics.wustl.edu
Fri Mar 16 22:02:26 EST 2001


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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