Hi everyone,

I'm having trouble getting protein expression with mRNA that I'm transfecting into my cells. I was wondering if mRNA secondary structure could be affecting things, so I inputted my mRNA sequence into ViennaRNA's RNAfold. However, I'm a bit lost with how to interpret the data.

RNAfold shows that my mRNA (1st picture) has a minimum free energy (MFE) of around -1500 kcal/mol and outputs a pretty scary picture with lots of secondary structures. However, when I input the sequence for human beta actin mRNA (2nd picture) the results also look pretty scary (it got -500 kcal/mol).

At what point is RNA secondary structure considered deleterious to transfection and protein expression? Since both my sample and beta actin have pretty high MFEs, maybe my mRNA isn't forming bad secondary structures?

Thank you!