Hello dear colleagues,
I want to overexpress a gene in zebrafish embryos and my strategy is to do in vitro transcription to make mRNA and then inject them into the embryos. However when I collect some embryos a few hours latter to check the overexpression by real time PCR, I dont see any difference between the injected and the non-injected subjects. I tried injecting more mRNA but i did not work either. What am I doing wrong? Is there any better strategy? Since nobody in my lab has experience with transgenic fish I am avoiding this option.
Thanks in advance!
Hello, did you check your mRNA has the elements required for translation and protection from endogenous RNases (cap, poly-A tail), if those elements are Ok, probably is degrading during the microinjection process, are all the materials RNAse free?
I don`t know your experiment design or your goals, but I think that if you are introducing mRNA you should analyze overexpression by western blot instead qRT-PCR
Analyzing the transcript is not a good assay because the transcript is what you are injecting. It is superior to analyze by Western Blot / ELISA to see if your mRNA is being translated into the protein of interest.
As recommended above, checking the construct sequence is important. After synthesis, check your product on a denaturing gel to see that the transcript is intact and the right size (not truncated). Also, after you inject, any RNA left over should be run on a similar gel to see that the transcript is not getting degraded during handling. If still in doubt, you might try a eukaryotic in vitro translation kit to see if your mRNA is making your protein. Good luck with your experiment.
You actually need another method as suggested by Elizabeth LeClair maybe It is superior to analyze by Western Blot / ELISA check this paper
Article Overexpression of Endothelin 1 Triggers Hepatocarcinogenesis...
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