Viromer® in mRNA transfection

mRNA transfection oversteps limitations of plasmid DNA transfection

Transfection of plasmid DNA is the easiest and the most common method to overexpress proteins in cells grown in culture. When it fails, the transfection reagent is generally recognized as the culprit, or the cells are simply considered as “hard-to-transfect”.

However, not DNA uptake/delivery, but rather its processing once inside the cell might be the major limitation of the technique. Reaching and penetrating the nucleus, transcription and release of mRNA to the cytosol before final translation into protein are all critical steps.

Transfecting cells with mRNA sequences rather than plasmid DNA constructs gives then a great chance to significantly increase transient protein expression levels in a majority of cell types, and offers a unique alternative for challenging cells.

mRNA is faster!

mRNA does not need to reach the nucleus for cellular action. Translation occurs through a promoter-independent process and the desired pro­tein is detectable as early as 6 h post-transfection.

mRNA is safer!

There is no risk of genomic integration and transient expression avoids toxicity related to protein accumulation.

mRNA is a very productive template

As simple as in nature, mRNA instructs directly cells to produce proteins. With an optimized delivery, it is then possible to reach very high expression levels of any intracellular, transmembrane or excreted protein.

Viromer® RED and Viromer® YELLOW have been designed to work equally strong with DNA and mRNA, even enabling their co-transfection. Based on internal tests and users’ feedbacks, we have demonstrated a clear advantage of mRNA transfection in general, but more interestingly for some specific cells known as “resistant” to plasmid transfection:

  • cells with low division rate, e.g. primary neurons, differentiated skeletal muscle cells, and
  • cells with cytosolic defense mechanisms against foreign DNA (innate immunity), e.g. AIM2-Interleukin or cGAS-Interferon enzymatic cascades of macrophages and monocytes.

We have successfully transfected mRNA into several model cell lines (CHO, HEK, HeLa, HepG2, C2C12, MDCK, HUVEC), but also in some specific cancer cells (A549, H322, H358, Neuro2A, SH-SH5Y), primary neurons, and immune MPS cells (primary macrophages, monocytes and dendritic cells, THP-1 monocytes, J774 macrophages). For more details, please go to the cell database.

Comparative transfection efficiency of Viromer® RED used for plasmid DNA or mRNA delivery in diverse immune cell lines (maximal percentage of positive cells reported by users).

How switching to mRNA transfection?

The efficiency of transfection and subsequent translation will strongly depend on the structure of the mRNA used for coding the desired protein. Recent advances on mRNA modified chemistries have permitted to synthetize stable (extended half-life) and high quality mRNA simulating wild eukaryotic mRNA, with a reduced immunogenicity.

For producing modified mRNA, we recommend to use in vitro transcription commercial kits enabling 5´ capping and 3´ polyadenylation, followed by purification of the tran­scribed mRNA. As template, any linearized plasmid coding the gene of interest (regulated by a T7 Polymerase promoter) or a PCR product are usable. An alternative to homemade in vitro transcribed mRNA is to purchase reporter or customized mRNA to an oligo supplier.

Our Start Positive® Controls easily guide you on the transition to the RNA world to maximize your results. For more details follow this link to the lyophilized in vitro reagtens. Just rehydrate the lyophilized powder with the provided buffer and transfect your cells with a GFP-encoding mRNA versus a GFP-encoding plasmid to figure out, if mRNA could be a new tool for your research.

Colors

Viromer® BLUE | Viromer® GREEN | Viromer® RED | Viromer® YELLOW

transfection efficiency

< 30% | 30-50% | 50-80% | > 80%

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Type siRNA mRNA pDNA Data
A549              See data Publication
C2C12               See data Publication
Cardiomyocytes, primary murine         See data Publications
CHO                    Publications
Dendritic cells, primary human and mouse                See data Publications
H322         
H358          
HEK-293 T                Publications
HeLa                  See data Publications
Hep G2               Publication
HUVEC, primary human umbilical vein endothelial cells               See data Publication
J774                See data
Macrophages, primary human and mouse                 See data Publications
MDCK                  See data
MDSC Myeloid derived suppressor cells        See data
Monocytes, primary human               See data Publications
Neuro-2A                       See data
Neurons, primary murine      See data Publication
RAW 264.7              See data Publications
SH-SY5Y              See data Publication