No expression from injected GFP&RFP mRNA - (Oct/31/2007 )
Thank you very much. I'll try that tomorrow.
I did it this afternoon. Six hour embryos were still dark.
I really have no idea now.
Anyway, thank you very much.
Thank you genehunter. The only thing that differ from others work might be an additional AgeI site plus several nucleotides (ACCGGTCGCCACC) before start code ATG. Do you think this could affect the expression? But if it does why I could see the fluorescence from the DNA injected embryos?
So many thanks to you!
And have a good weekend.
I am not sure. I still think the cap structure is something that needs to be looked upon.
Adding 5' UTR will be my another suggestion. Some expression vectors allow you to co-express more than one genes from the same enhancer using an "internal ribosome entery sequences". Clone this sequence 5' of your coding sequence, make mRNA from it, see if that helps.
I remember the kit manual said the using of restriction enzymes which give 3' overhanging ends will cause a low level transcription. Do you think this will affect?
But I did get enough quantity of mRNA checked by both gel and spectrophotometer.
You would expect the questionable sequence is present in cell-expressed mRNA, dont you? You clearly got good expression from that.
OK, so I know this is an old post but I was glad to see I am not the only person in the world with this problem!
I have had this problem for the past year, making any functional experiment extremely difficult.
Maybe you have solved your problem (or given up), but just in case, here is what I have figured out....
1. Sometimes, a random "toxin" from your original plasmid prep can survive through the digest, gel purification, transcription, and RNA purification/precipitation. I have found that doing a phenol-chloroform/precipitation of your original plasmid can solve most problems.
2. 3' overhangs DO matter! The Message Machine kit says that there can be accidental transcription of the other strand if your template is linearized this way. That means that you are effectively injecting double-stranded RNA. In zebrafish this can be REALLY bad! In her post-doc, my supervisor wrote a paper on how RNA interference has non-specific effects in zebrafish. That means that not only your RNA, but other RNAs can be degraded.
3. You seem to be injecting GFP at a really high concentration! We typically inject at a final concentration of 35-50ng/microlitre for yolk injections. Weird/unexplainable things happen when you inject high concentrations of RNA.
Does anyone else have this problem? Other suggestions?