https://www.researchgate.net/post/How_to_do_a_rescue_experiment_in_14dpf_Zebrafish

Hey Oscar,

We recently established a method for transfection with the JetMessenger transfection reagent and in vitro transcribed and immunologically silenced mRNA. This method was used for in vitro transfection of cells so far, but it also describes very detailed how your mRNA should be designed and prepared before usage.

Modifications during mRNA transcribtion were:

Removing any traces of DNA templates (because this induces death of macrophages and other phagocytic immune cells).

Use of 5-methyl-CTP and pseudo-UTP, which are bases that reduce immunogenicity and enhance stability of the mRNA.

Dephosphorylation of the 5’-ends of the in vitro transcribed mRNA by Antarctic phosphatase to prevent recognition by the RIG-I complex.

For a detailed protocol (including primer desing, kits of choice and purification of mRNA) please have a look at:

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA. J. Vis. Exp. (Pending Publication), e60143, In-press (2019).

All the best,

Marc

Hi, Oscar

Does RNA sequence contain any target protein? Or it's just a template for enhancer or promoter? If it contains your target protein, you can just start from ATG or 6 codes before ATG. After the stop code, you need to add some poly(A) tail to slow down the degradation.

It depends on your design of your study. For signal pathway, we will add another activated or deactivated form of RNA sequences as positive or negative control to compare with the full-length of mRNA.

For enhancer or promoter, these genes can regulate the gene expression. Then, you might need to try different sequences to confirm the specific codes that regulate the protein expression.

Good luck.

Yihui

Yihui Huang Thank you for your answer. Yes it contains my target protein. I have designed primers from the beginning and the end of the protein(20bp for each), but the Tm is 51C and 42 C.

How to solve the Tm problem? Should I design a longer primer?

There are some tips for PCR.

1. The best Tm is around 52 to 58. I usually test different Tm from 50 to 57 degree for the best condition (eg. more amount of mRNA, less non-specific bands). The problem is that the lower Tm you get, the more non-specific target sequences are amplified. Even if the Tm are different (The difference shouldn't be more 7 degree.) between left and right primers, it's still okay to use the same temperature for PCR.

2. Try to add more CG than TA in the sequence. But in most of cases, Tm will still be less 45 degree but you can try to make it close to 45. Make sure you don't count the poly (A) tail into the Tm. And try to make both Tm for left and right primers as close to 52 degree as possible.

3. Use those polymerase that can proof read the sequence for PCR.

4. The range 18bp to 28bp is acceptable for primer designs.

I hope these helps.

Best Regards,

Yihui