antisense oligonucleotides

M K Bennett pamkb at mail.bris.ac.uk
Wed Mar 22 04:08:44 EST 1995


Jan Eggermont (Jan.Eggermont at med.kuleuven.ac.be) wrote:
: Hello all,

: I am planning to use antisense oligonucleotides to silence the expression
: of a gene in permanent adherent cell line. In view of this I would greatly
: appreciate your input on:
: 1) the chemistry of oligo: ordinary or phosphoro-thioates

Ordinary oligos get degraded very quickly especially if there is serum
present in the media.  I would try phophoro-thioates first.

: 2) the target of the oligo in the mRNAe: can you have antisense oligo's
: against any part of the mRNA or are preferential target sites for
: anti-sense oligo's.

Best to target around the start site of translation.  Some people have
reported that oligos targeted downstream of the start site work but it
appears to be really dependant on which system you happen to be studying
at the time.

: 3) effective concentration range of oligo's and duration of treatment to
: get maximal inhibition

You need to work this one out for yourself try around 2.5uM in the media
as a starting point.

: 4) which control oligonucleotides to use

Try random oligos, try oligos that are the opposite in sequence to yours
and importantly try just using dNTP's at a similer concentration to that
which would occur if your oligos were completely digested.


mike

-----------------------------------------------------------------------------
Mike Bennett D.Phil		|Scientists have odious manners, except when
Dept Pathology and Microbiology,|you prop up their theory; then you can borrow
University of Bristol, UK	|money from them--Mark Twain 1917 
m.k.bennett at bristol.ac.uk	|
-----------------------------------------------------------------------------





More information about the Methods mailing list