dsRNA Soaking

CHAD A. RAPPLEYE rappleye at biomail.ucsd.edu
Mon Aug 2 14:59:36 EST 1999


Hi Chris,

While I have not done extensive work with soaking (I prefer injections) I
have had success with the following:

1.  Pick 20-50 L4 hermaphrodites into RNase-free water in eppendorf tube
2.  Wash worms 3x in RNase-free water
3.  "suspend worms in ~5 ul of dsRNA solution
4.  incubate 10 hrs to overnight at 20 degrees
5.  transfer worms to OP50-seeded plates for >12 hours to allow further
development to adult stage
6.  examine progeny for phenotypes at increasing time intervals (different
RNAs seem to have different "onset times"

As a positive control for dsRNA effects, we have begun co-injecting unc-22
dsRNA with our experimental RNAs (unc-22 RNAi as described in A. Fire, et
al.  1998  Nature 391:806-811).  unc-22 RNAi is easily scored, non-lethal,
and has early onset in the F1 progeny.  Of course if you're looking for
Unc phenotypes, obviously unc-22 is an inappropriate control.

Good luck and don't give up on injection yet,


Chad Rappleye
Aroian Lab, UC San Diego



On Fri, 30 Jul 1999, CHRIS.J.MEE wrote:

>
> As a first year PhD student who is currently working with dsRNA
> interference in C.elegans i was interested to see in some of the
> abstracts at the 1999 International Worm Meeting that a number of
> people were using dsRNA soaking as a method of RNAi instead of
> microinjection.
> I dont seem to be able to find anything further on the technique so i
> was hopeing that someone may be able to fill me in on how its done
> and how successful it is compared to microinjection of dsRNA.
> I would also like to know if anybody has a good gene to use as a
> positive control in dsRNA injections as the genes i am looking at
> have not been used before and so i have no idea if they give an
> obvious phenotype. It may be that my injection technique needs work
> so a positive control with a good phenotype would help.
>
> Any help would be great
> Chris Mee
> University of Nottingham
> plxcjm at nottingham.ac.uk
> ---
>
>
>





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