No expression from injected GFP&RFP mRNA - (Oct/31/2007 )

I used just GFP and RFP mRNAs, which were transcribed by engineered pCS2+ plasmids (GFP and RFP coding sequence into BamHI&XbaI digested pCS2+) using ambion mMessage mMachine Kit and further purified by MEGAclear and eluted using elution buffer provided in the Kit. Then injected at the concentration of 200ng/ul, 400ng/ul and 800ng/ul into one-cell staged WT zebrafish embryos. But I can't see any fluorescence from 16 and 24 hpf embrys.
I'm really eager to solve this problem, for both the constructs sequence and the RNA quality are really good, and now I really don't know where the problem is.
Anticipating your answers!
Thanks very much.
Wei

Have you included 5'-Cap struture or 5'-UTR to enhance translation?

Thank you genehunter! The mMessage mMachine kit generated m7G(5')ppp(5')G capped mRNA and I used the 5'UTR on the vector.
Thanks again.

Has the mRNA been verified as functional by transfection on cells in vitro?

You would predict that the DNA template should be functional by microinjection as well, right? Have you check that?

Have you also used dye-dextran as an indicator for microinjection technique?

Thanks very much.
Actually I used the same sequence as other people used in many papers. I mean here the same GFP sequence in the same pCS2+ vector (also I used the same cloning site), besides this I generated the pDsRed control mRNA (I used them as a control)
. Yes, I should test whether the mRNA is functional by what you suggested but we don't have the zebrafish cell line here. But why if they still not work in cells? I just planned to microinject the DNA template tomorrow morning and I will let you know when I get the results this Friday. And I did use dye but here was phenol-red.
Thanks again.


Has the mRNA been verified as functional by transfection on cells in vitro?

You would predict that the DNA template should be functional by microinjection as well, right? Have you check that?

Have you also used dye-dextran as an indicator for microinjection technique?
[/quote]

I microinjected the SacII linearized plasmids (the same for the in vito transcription template) and I did see strong mosaic fluorescences from all all of them. So I think now maybe the cap adding reagent was not working then I got mRNAs without 5'cap. I'll change the Kit and see what will happen. And what's your suggestions?
Thanks.
Wei

You may want to discuss with the provider about this issue, see what they have to say. I know NEB has the cap structure.

It's really a shame. I tried a new transcription kit but I still got no fluorescences(three different concentrations of 200, 400, and 800ng/ul).
I checked again the RNA by running a gel (Normal TAE buffer and agrose). I loaded the sample at the quantity of 200ng and 600ng. I saw a clear band from 200ng but lots of smear (Smaller than the main band in size, may be the degradation?) from the 600ng one.
Do you think this could affect? I also checked the phenol red I used to dye my sample, but it didn't look like could degrade my samples. I have tried all what I can know to do but still can't find the reason. I'm really frustrated now.
Thanks for time.
Wei

I recall one paper reported much earlier peak expression onset, as early as 6 hrs. I assume the decade will be faster thna DNA-based expression as well. So check at different time points to see if that gives you any luck.