FAQ: mRNA transfection

jetMESSENGER® protocol is simple: the reagent is ready-to-use and is compatible with the standard growth media (with or without serum and antibiotics) of a wide variety of adherent and suspension cell types. jetMESSENGER® operates through a very gentle process and upholds cell viability and overall cellular morphology in a variety of hard to transfect cells, such as rat cortex neurons, human liver carcinoma cells and mouse embryonic stem cells.

The success of mRNA delivery is mainly due to circumventing the need for nuclear import of DNA, hindered in slow- to non-dividing cells. Therefore, by switching to mRNA transfection, cells that are slow-dividing or that have developed specific mechanisms to protect their genome can finally be used for gene expression, with unmatched transfection efficiencies.

mRNA can be produced using commercially available solutions (e.g. HiScribe™ kit provided by New England Biolabs) or be purchased “custom made” through different oligonucleotide suppliers such as Trilink, Eurofins or Aldevron.

mRNA is synthesized from DNA templates such as plasmid DNA, PCR product, synthetic oligonucleotides. Importantly, the template must be linearized and contain a RNA polymerase promoter region such as T7 or SP6 upstream of the sequence to be transcribed, in 5’ for the sense RNA or in 3’ for the antisense RNA. If required, the RNA polymerase promoter region can be added upstream of the sequence to be transcribed by PCR using primers containing the minimal promoter sequences.

Learn more about mRNA synthesis:

mRNA Synthesis

Generally speaking, mRNA purification following in vitro transcription is not necessary, as there should be minimal RNA contaminants. If needed, several methods are suitable, including Phenol-chloroform Extraction/Ethanol Precipitation, LiCl precipitation, gel purification methods, and several commercially available mRNA isolation kits.

There is no limit in mRNA size per se. It is worth to keep in mind that mRNA synthesis kits are usually optimized for mRNA transcripts ranging in size from 0.3 to 5kb, equivalent to a protein size of 10-150 kDa. Therefore, for shorter or longer mRNA transcripts, additional optimization may be required to obtain a similar high yield.

In comparison to DNA transfection, mRNA-mediated transfection with jetMESSENGER® enables a more controlled gene expression that is not promoter dependent, over a similar period of time. In actively dividing cells, mRNA expression will be more prominently and stably observed between 1 to 4 days post-transfection. In slow to non-dividing cells, mRNA expression can be stable over a longer period of time, as for example up to 7 days in primary neuronal cells.

During the synthesis of endogenous eukaryotic mRNA, a 7-methylguanosine CAP is added in 5’ on the nascent transcript to protect it from degradation and increase translation rate. The presence of a polyA-tail in 3’ of nascent mRNA further protects from degradation and plays a role in the nuclear export of the mature mRNA. Therefore, existing mRNA synthesis kits usually offer solutions to improve stability of mRNAs by incorporation of 5’ cap analog, and 3’ polyA-tail. In addition, specific base modifications within the mRNA sequence such as 5-methylcytosine and/or N6-methyladenine can be incorporated for increasing stability and decreasing immunogenicity. Custom-made mRNA ordered from an oligonucleotide provider can be ordered with specific modifications such as Pseudo-Uridine for increased stability.

Increasing the stability of the RNA transcript may not always be necessary. For example, some viral RNA transcripts do not need to be capped and polyadenylated in order to start replicating and/or produce infectious virus after transfection.

mRNA delivery is suited for transient expression, but not for the generation of stable clones. mRNA cannot integrate into the cell genome, which prohibits the generation of stable clones but has the advantage of not modifying the genome of the host cell.