Help Help with microinjecting RNA

[cheuk...@gmail.com]

Hi all,

I am new to microinjection of zebrafish embryos and I need help.

Briefly, I have injected 200pg of capped EGFP-coding RNA and the
embryos show no expression. In addition, their yolk kind of leaks out/
burst between 4hpf and 10hpf, thus killing the embryo.While uninjected
ones look fine.

To help with understanding the problem, I have detailed my protocol
here.

Capped RNA Synthesis

I linearized pSP64TNE-EGFP at the Sal I site, Phenyl-Chloroform
extracted the linearized template and then transcribe capped RNA with
SP6 promoter using the Ambion mMessege Kit. The yield comes in at an
acceptable 21ug with A260/280 at 1.94 and a A260/230 at 2.36. Running
200ng of the capped RNA in a 1% agarose gel gives me a sharp single
band.

Microinjection

I diluted the capped RNA to 50ng/ul in 0.1M KCl and 0.05% PhenolRed.
4nl(200pg) was injected into each embryo, the embryos are recovered
into 30% Danieau's Solution, and keep at 28.5C incubator.

Any idea what can I be doing wrong here?

Thanks a million for reading.

Tony

[Bruce@WHOI]

Tony-
I spoke with Matt Jenny, a post Doc in our lab, who has been using the
techniques you are attempting, and he says:
4 nL is OK but I would not go any higher....2 nL is better. The
location of microinjection is critical....need to be in the cell, or
if in the yolk very close to the cell. Do not use 0.1M KCl. I
microinject 2 nL with the mRNA in RNase free water with 0.001% phenol
red. The KCl may be throwing off the osmotic homeostasis.....my guess
is the eggs/yolk is swelling because of the influx of water. Also,
with that amount of eGFP you should be able to detect translation as
early as 3-4 hours post fertilization and the 24hpf embryo should
light up like a christmas tree.
Cheers,
Bruce

On Oct 22, 12:20 am, "cheuk.ch...@gmail.com" <cheuk.ch...@gmail.com>
wrote: