[Zbrafish] Re: Help with microinjected mRNA

[Tony Cheuk-Chung Ho]

Wei,

After reading Bruce's advise (refer to my previous post
http://groups.google.com/group/bionet.organisms.zebrafish/browse_thread/thread/5c2bd01c334f425a),
I precipitate my capped RNA and resuspend in RNase-free water. At this
point, I tried to make them as concentrated as possible, maybe around
500ng/uL. Before injection, I mix the 500ng/uL stock with 0.05% Phenol
Red in RNase-free water. Then, I inject embryos at 1-2 cell stage with
0.5, 1nL, and 2nL. This seems to be working fine for me as expression
goes. Though not as "christmas tree" as I have wished, but expression
is crisp and strong at 24hpf. I was able to observe weak fluorescence
signal as early as 4-5hpf.

Hope it helps.

Tony


On Nov 9, 2:46 am, wei xia <x... from umbi.umd.edu> wrote:
> Hi Tony,
> Sorry for the bothering to other peoples. I have the same problem you
> had before. I injected in vitro transcribed capped GFP mRNA (Ambio
> mMessage Kit) into zebrafish 2-cell-stage embryos but I got no
> expression from it. Instead when  I injected the plasmid templates
> which I used for the transcription I got strong fluorescence. I saw
> Bruce's reply and he said " 24hpf embryo should light up like a
> christmas tree." But why I couldn't!
> How could you solve your problems?
> Any suggestions are very very great appreciated!
> Wei
>
> Following is your post.
> 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