Table 8 |.
Troubleshooting table
| Step | Problem | Possible reason | Solution |
|---|---|---|---|
| 26A(iii), 26B(iii), 26C(iv) | mRNA degradation | mRNA is susceptible to degradation by introduced exogenous RNase during operation | Use RNase-free reagents, pipette tips and tubes. Clean the working bench before operation |
| 26A(iv), 26B(v), 26C(viii) | The concentrations of components in the formulation are not correct | The lipid mix solution or mRNA aqueous solution is not mixed well | Vortex and centrifuge tubes of lipid mix solution and mRNA aqueous solution before mixing the two phases together to ensure correct concentrations |
| Ethanol has evaporated, thereby increasing the concentration of lipid stock solution and affecting the formulation ratios | Remake stock solutions from scratch | ||
| 26A(iv) | The two phases do not mix well in the pipette mixing method | The hand pipetting is not rapid | Rapidly pipette up and down immediately after adding the aqueous solution into the ethanol solution |
| 26B(iv) | The vortexing is not at a moderate speed | A different type of vortex mixer is used. For a Vortex-Genie 2 vortex mixer, speed level ‘1’ is appropriate. However, for a Fisher Scientific vortex mixer, levels ‘4’-’5’ can reach a similar speed | Adjust the vortex mixer to an optimal speed level |
| 26B(v) | The two phases do not mix well in the vortex mixing method | The addition of the lipid ethanol mixture solution into the vortexing solution is not a single action | Carefully insert the pipette to the bottom of the tube, and quickly add the ethanol solution in a single action |
| 26C(viii) | The two phases do not mix well in the microfluidic mixing method | The cartridge is not washed after each formulation run. The cartridge is overused and blocked | Use deionized water and ethanol to wash the cartridge after each formulation run. Change to a new cartridge after using it for over five formulations |
| 40 | No detectable signal after injection of luciferin | Intraperitoneal injection of d-luciferin is not successful | Avoid subcutaneous or muscular injections |
| Box 1, step 2 | DOPE does not dissolve very well in ethanol | DOPE has limited solubility in ethanol | Heat the solution slightly to increase the solubility: immerse the glass vial into a 50 °C water bath for a few seconds, and then gently shake it; repeat the process until the solution appears clear. The vial should be sealed to avoid ethanol evaporation |
| Box 1, step 7; Box 2, step 6 | A white precipitate of 18PA is observed in the solution | The stock solution of 18PA is not properly stored and THF evaporates | Store the 18PA stock solution at −20 °C, and put the solution on ice while handling it to decrease the THF evaporation. The best way is to make the fresh stock solution each time and use it right away |