stealth siRNA optimisation

Jayakumar, R via methods%40net.bio.net (by R.Jayakumar from roswellpark.org)
Fri Jul 11 15:22:25 EST 2008


Depends a lot on the cell line and amaxa protocol.  You can keep
increasing it till you see an optimum between cell death and efficiency
in knockdown.
Jay
 
-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu
[mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Scott Brown
Sent: Friday, July 11, 2008 5:28 AM
To: methods from magpie.bio.indiana.edu
Subject: stealth siRNA optimisation

Hi,
 
I have been trying to use some invitrogen stealth siRNA to knock down
the function of the glucocorticoid receptor and expression of BCDIN3. 
I am using a nucelofector for transfecting the siRNA, it recommends
using 2uM [siRNA], I have tried using 5um and 10uM as well and have
achieved better results (almost 80% knockdown).
 
I always thought high concentraions of siRNA can be toxic to cells, can
anyone advice on the mechanism of action responsible for this toxicity,
is their any limit to how much siRNA i can expose my cell line to or is
this a typical scenario when optimising siRNA knockdown.
 
Thanks
 
Scott Brown
PhD Candidate
Children's Cancer Institute of Australia

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