The requirements for using CleanCap mRNA are no different than using ARCA mRNA. You can use your standard electroporation or transfection conditions. There are several advantages with using a CleanCap mRNA. The translational efficiency far exceeds that of a standard ARCA capped mRNA. You may want to re-purpose your solar eclipse glasses when viewing your cells after EGFP translation!
And just like with ARCA, you can submit your open reading frame to TriLink for custom mRNA transcription and we will do the rest!
There are typically two avenues for transfection of mRNA depending on cell type:
1. For adherent cells, use a transfection reagent such as MessengerMax (Invitrogen), mRNA TransIT (Mirus) or mRNA-In (MTI-GlobalStem).
a. For a 24-well plate format, use a ratio of 500 ng mRNA:1 ul transfection reagent in a total volume of 50 ul complexed mRNA/lipid per well. This is a good starting point and is described in this protocol. Diluting your working stock of mRNA down to 100 ng/ul works well to establish manageable volumes for mastermixes.
b. We’ve only used TransIT and mRNA-In in-house – from our experience these reagents give a dose response of FLuc activity from between 100 ng to 400 ng. Therefore, a minimum starting amount of 100 ng is sufficient. Use 100 ng mRNA: 1ul reagent in a complexed final volume of 50 ul per well.
c. Ratios for other plate formats need to be optimized according to the manufacturer’s instructions.
2. For cells in suspension,
such as CD34+ cells, electroporation has traditionally been the mode of mRNA delivery. However, transfection reagents have advanced and some are able to transfect cells in suspension reliably, such as the transfection reagent mRNA-In (MTI-GlobalStem).
a. For electroporation, an instrument from Lonza is recommended based on advice from a trusted collaborator.
b. Use 1-15 ug of mRNA per million cells in a 100 ul volume.
In general, 100 ng/uL is not considered a high concentration. We supply our mRNA at 1 mg/mL in 10 mM Tris-HCl, pH 7.5. That said, the mRNA sequence and structure can dictate its solubility. We recommend heating the mRNA for 15 min at 37°C to improve solubility. Long incubations at elevated temperatures should be avoided.
In regards to the diminishing activity of your mRNA, have you checked for degradation? In addition to the solubility, your issues with mRNA activity may also result from RNA degradation over time. To combat degradation you should use RNase-free reagents and materials and use proper technique. Additionally, we suggest that you aliquot your RNA to limit freeze/thaw cycles. A higher concentration may also improve stability however could exacerbate your issues with solubility.
Please let us know if we can help you further.
mRNA offers several advantages over traditional plasmid and viral-based approaches:
- mRNA boasts a superior safety profile. As a transient carrier of genetic information, it is metabolized naturally and poses little to no risk of genomic integration. Additionally, no inactivated viruses or pathogens are needed.
- mRNA serves the dual purpose of expressing the desired antigen as well as acting as an adjuvant.
- mRNA triggers a more diverse immune response. Because the mRNA encoded epitopes are intracellular, they are recognized by the immune system in an MHC class-independent manner.
- mRNA can more readily transfect difficult-to-transfect cell types because it functions in the cytoplasm. DNA vaccines can be limited by lack of access to the nucleus.
- mRNA manufacturing is easily scalable. Because mRNA transcription is carried out completely in vitro, to hundreds of millions of vaccine doses with a lead time of as little as a few weeks. This allows for rapid deployment of a new antigen during pandemics.
- mRNA is easily customizable. The ease of manufacturing makes it a viable option for personalized treatments.