Hello at all,
Inspired through a paper that used the SuperFect Transfection Reagent to transfect siRNA to A431 cells I purchased it myself with the aim to transfect eGFP mRNA to HEK 293 cells.
I performed the transfection according to the manual and tried different concentrations of mRNA and superfect as suggested. The only difference was that the IVT-generated mRNA was eluted in RNAse free water instead of TE-buffer.
Unfortunately, none of my transfection approaches worked at all.
Now I’d like to ask if you know any specific reasons why the transfection of mRNA with SuperFect wouldn’t work.
Thank you in advance.
Several factors could be responsible, from the siRNA through the reagents used to the method of getting your siRNA into the cells and others. The first question is 1. Are your siRNA specific for the gene(s) you are targetting? 2. Is your method of introducing the siRNA into the cells good enough 3. Did you check for the uptake of the siRNA by your cells and so on. Until you detail what you have done, it is difficult to tell where you go wrong
Hello,
I would simply suggest to use another reagent... First Superfect is designed for DNA transfection. If you report that it was used for siRNA, why not, but be careful with mRNA, it's not the same. Usually, reagents designed for siRNA do not work for large RNA like mRNA.
As an example and to have more info, you can have a look to our Viromer RED, this reagent is made for that purpose. At your disposal if you have further questions. Bests, Sandra
https://viromer-transfection.com/mrna-transfection
Sirna and mRNA cannot be transfected the same way. Being double stranded sirna is more stable than highly folded mRNA. mRNA or sirna firstly enters the nucleus if ur mRNA doesn't have the 5 cap or poly a tailing they would never come out .. however sirna are processed in the nucleus and destined to export to the cyto where it actually works. So dividing cells are preferred for sirna where as non dividing cells are preferred for mRNA. Overall using gfp rna as positive control for sirna is not a good option. Why Don u go for oligo cloning in appropriate expression vector with gfp reporter system. I even doubt if ur in vitro transcription worked on the whole sequence. Single base loss will terminate it's expression. If u still want to use it make sure it's full by amplifying the ends
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