[Zbrafish] Help with microinjected mRNA

[wei xia]

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
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