hello,
i am planning to transfect mouse primary hepatocytes with miRNA in order to isolate microsomes to study the activity of my enzyme of interest.
Therefore, I wondered if the primary hepatocytes will survive an 24 h incubation in a serum-reduced Opti-MEM/Lipofectamine 2000 transfection solution since primary hepatocytes are very sensitive and must be cultured in medium containing serum. In order to ensure transfection of miRNA, the medium must not contain serum. That´s why I´m running in a little discrepancy !!! Does someone have experience with transfection of miRNA into mouse primary hepatocytes using Lipofectamine 2000/Opti-MEM solution and can tell me whether I can incubate hepatocytes in a serum-reduced environment for 24 h ?
Thank you all for your efforts to answer my question.
Best regards
Stefan
Dear Stefan,
This is true only for Lipofectamine 2000, there are many other possible reagents on the market and I am sure this one is not the optimal for primary hepatocytes (even within the Lipofectamine family)!
But why not using another technology like our polymer-based Viromer transfection reagents. They work with serum and any kinds of media.
and they are made in Germany ;-)
I do not have a reference for miRNA in hepatocytes, but please check the 2 following articles. They cite use of Viromer in mouse primary hepatocytes, for siRNA with Viromer BLUE, for plasmid with Viromer RED. In both cases, hepatocytes were cultivated with serum. And the medium changed after transfection.
Article Hepatic Fgf21 Expression Is Repressed after Simvastatin Trea...
Article Identification of Insulin-Responsive Transcription Factors R...
if you cannot access the pdf files, please write me, I can forward the Materials and methods to you.
In your case, you would need the Viromer BLUE reagent. Same thing, if you want a free sample to try, just let me know.
Best,
Sandra
Hi Stefan
Mouse primary hepatocytes can be quite hard to transfect in my experience. You may end up with low efficiency.
A better approach would be to use a lentivirus, which will transduce primary hepatocytes with high efficiency with stable effect. Lentiviruses have been used widely for miRNA expression with good results. I’m happy to advise on strategies for this.
Best wishes
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