FAQ

N1-methyl-pseudouridine (m1Ψ) is a modified nucleoside that plays several key roles when incorporated into mRNA:

• Enhanced Stability: mRNA with m1Ψ modifications demonstrates increased stability, prolonging the half-life and allowing for longer-lasting protein expression
• Reduced Immunogenicity: m1Ψ reduces the immune response typically triggered by unmodified mRNA, preventing inflammation and degradation, making it more efficient for therapeutic delivery
• Improved Translation Efficiency: m1Ψ enhances translation efficiency by promoting ribosome binding and the initiation of protein synthesis, resulting in higher protein output
• Resistance to RNA Editing Enzymes: m1Ψ provides protection against RNA editing enzymes like ADAR, which can alter the mRNA sequence by editing adenosine residues. This ensures the integrity and fidelity of the mRNA sequence during protein synthesis.

To assess size-based purity, we run the mRNA sample through a bioanalyzer. The purity is determined by calculating the ratio of the main peak (target size ±15%) relative to the total signal.

Residual double-stranded RNA (dsRNA) can form during the in vitro transcription process used to synthesize mRNA, potentially triggering unwanted immune responses in humans. To detect and quantify trace amounts of dsRNA contaminants, PackGene uses a double-antibody sandwich ELISA.

The process starts with coating the wells of an ELISA plate with a monoclonal or polyclonal antibody specific to dsRNA. When the mRNA sample is added, any dsRNA present binds to the capture antibody. After incubation, unbound material is washed away, and a second, labeled antibody—also specific to dsRNA—is introduced. This second antibody attaches to the bound dsRNA, forming a “sandwich” complex.

The detection antibody is typically linked to an enzyme, such as horseradish peroxidase (HRP), which reacts with a substrate to produce a measurable signal (colorimetric, fluorescent, or luminescent). The intensity of the signal is proportional to the dsRNA concentration and is quantified using a microplate reader, with values compared to a standard curve of known dsRNA concentrations. This highly specific and sensitive method ensures the safety and purity of mRNA therapeutics by detecting low levels of dsRNA contamination.

Our standard mRNA purification protocol is optimized to reduce dsRNA to very low levels, with final products containing less than 0.1% dsRNA (verified by ELISA). If a more stringent dsRNA threshold is required, we offer additional methods to further reduce dsRNA during mRNA production. Please consult with our technical support team for more details.

For RUO-grade customized mRNA, we use LiCl precipitation for quantities under 1 mg. For larger amounts (>1 mg) or off-the-shelf mRNA, we use HPLC-based Oligo dT purification to enhance RNA purity.

Our standard mRNA concentration in LNP is 0.2 mg/mL, but we can increase this to 2 mg/mL. If higher concentrations are needed, please contact our technical support team.

mRNA-LNP can be stored at -80°C for up to 2 years. However, mRNA-LNP is highly sensitive to freeze-thaw cycles, which can compromise its ability to transduce cells or tissues. To avoid this, please inform us of your preferred aliquot sizes. Our pre-aliquoted mRNA-LNP kits come in volumes of 10 µL or 50 µL.

mRNA can be stable for at least 2 years when stored properly at -80°C and if repeated freeze-thaw cycles are avoided. Stability can vary based on the mRNA length, but we’ve tested mRNA to remain stable through 30 freeze-thaw cycles. For long-term storage, -80°C is ideal, while -20°C works for short-term storage.