Frequently Asked Questions:

TransIT®-mRNA Transfection Kit

The TransIT®-mRNA Transfection Kit provides high efficiency and low toxicity transfection of single-stranded RNA, including mRNA and viral RNA, into a broad spectrum of cell types. Transfections are most effective when carried out in complete growth media, with no media change or serum addition required. These unique features make the TransIT®-mRNA Transfection Kit ideal for in vitro RNA delivery studies.

GENERAL QUESTIONS AND ANSWERS

Q1. What is the difference between mRNA and an in vitro transcript?
mRNA is a single stranded RNA molecule that encodes a protein and may have post-transcriptional modifications such as a 5’ cap and 3’ poly(A) tail. It can occur naturally in a mammalian cell or be produced in vitro to mimic natural mRNA. An in vitro transcript is an unmodified RNA molecule produced from a DNA template using one of the three phage DNA-dependent RNA polymerases (T7, T3, or SP6).

Q2. What cell types have been successfully transfected with the TransIT®-mRNA Transfection Kit?
We have successfully transfected A549, CHO-K1, COS-7, HEK 293, HeLa, HepG2, NIH 3T3, and Vero cell lines. Additional cell types transfected with TransIT®-mRNA Kit can be found in our Citation Database.

Q3. The TransIT®-mRNA Transfection Kit contains two components, the TransIT®-mRNA Reagent and mRNA Boost Reagent. What is the formulation of each component?
The TransIT®-mRNA Reagent is a novel cationic polymer/lipid formulation and is non-liposomal. The mRNA Boost Reagent is a proprietary compound. Both components are required to complex with RNA for efficient delivery into the cytoplasm.

Q4. What are the recommendations for RNA production for transfection?
We routinely use the mMESSAGE mMACHINE T7 Ultra Kit (Ambion) to generate the in vitro transcripts (RNA) for transfection. This kit caps and polyadenylates the transcript so that it will more closely resemble mammalian messenger RNA (mRNA) and imparts efficient translation and stability. Subsequently, the in vitro transcribed mRNA is purified using the RNeasy® Mini Kit (Qiagen). Other in vitro transcription kits may be used to produce high quality RNA for transfection.

Q5. Is it necessary to cap and polyadenylate the in vitro transcript before transfection?
Capping and polyadenylation will increase both the level of expression and the intracellular stability of the in vitro transcript but it may not be necessary depending on the experiment. Alternatively, the in vitro transcript can be produced with an internal ribosome entry site (IRES) in the 5’ untranslated region of the mRNA. The presence of the IRES can increase transcription in the absence of a 5’ cap.

Q6. What sizes of RNA can be delivered using the TransIT®-mRNA Transfection Kit?
We have transfected RNA ranging from 0.94 - 3.2 Kb in size. Researchers at Loyola University Chicago have successfully delivered a 32 Kb viral RNA using the TransIT®-mRNA Transfection Kit.

Q7. Can the TransIT®-mRNA Transfection Kit be used to transfect suspension cells?
Yes. For suspension cells, we recommend plating cells at a density of 6–10 × 105 cells/ml.

Q8. How should I store the TransIT®-mRNA Transfection Kit?
Both the TransIT®-mRNA Reagent and mRNA Boost Reagent should be stored tightly capped at 4°C to prevent evaporation. Prior to use, warm both reagents to room temperature and gently vortex to dissolve any precipitate that may have formed.

Q9. Where can I find references in which other researchers have used the TransIT®-mRNA Transfection Kit?
The most recent updates are available in our Citation Database.

Q10. Can I use the TransIT®-mRNA Transfection Kit for siRNA delivery?
No. We recommend using either TransIT-TKO® or TransIT-siQUEST® Transfection Reagent for siRNA delivery.

PROTOCOL QUESTIONS AND ANSWERS

Q11. When using TransIT®-mRNA Transfection Kit, should complexes be added to cells in serum-free media or serum-containing media?
The highest transfection efficiencies are achieved when the complexes are added to cells in complete growth media (serum-containing). For clarity, form transfection complexes in serum-free media, e.g. Opti-MEM® I Reduced-Serum Medium, as serum can interfere with complex formation.

Q12. Do I have to perform a media change after transfection with the TransIT®-mRNA Transfection Kit?
When using the TransIT®-mRNA Transfection Kit, a post-transfection media change is not necessary. If your experimental set up requires a media change, we recommend waiting four or more hours following the addition of transfection complexes.

Q13. Will antibiotics in the culture media interfere with transfection efficiency?
Low levels of antibiotics (100X stock of penicillin/streptomycin/amphotericin diluted to 0.1-1X final concentration) in complete growth media do not interfere with RNA transfection using the TransIT®-mRNA Transfection Kit. However, use of antibiotics should be avoided at the transfection complex formation step.

Q14. After transfection using the TransIT®-mRNA Transfection Kit, what is the optimal time to assay cells?
The optimal incubation time will vary depending on the experiment and can be determined by testing a range from 4 - 48 hours.

Q15. Can I cotransfect two RNA transcripts using TransIT®-mRNA Transfection Kit?
Yes. The ratio of both the TransIT®-mRNA Reagent and mRNA Boost to total amount of RNA should be maintained.

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q16. How can I improve transfection efficiency using the TransIT®-mRNA Transfection Kit?
Multiple factors could contribute to low transfection efficiency; refer to the following critical troubleshooting tips to improve your transfection results:

Volume of TransIT®-mRNA and mRNA Boost Reagents - Determine the optimal amount of TransIT®-mRNA and mRNA Boost Reagents by titrating each reagent from 1-3 μl per well of a 12-well plate.
Amount of RNA - Determine the best RNA amount by titrating from 1-3 μg of RNA per well of a 12-well plate. For certain applications, the optimal amount may be outside of the recommended range. As a starting point, use 1 μg of RNA per well of a 12-well plate.
Transfection complex formation - Do not let this step exceed 5 minutes as longer incubation times may result in lower transfection efficiency.
Quality of RNA - RNA used for transfection should be highly purified and sterile. We recommend purifying the RNA using a column procedure such as RNeasy® spin columns (Qiagen). Avoid RNase contamination as degradation of the RNA substrate will significantly diminish expression of the transfected RNA.
Cell density (% confluence) at time of transfection - Determine the optimal cell density, which may be outside the recommended range of 60-90% confluence, in order to maximize transfection efficiency.
Inhibitors of transfection - The presence of polyanions, such as dextran sulfate or heparin, can inhibit transfection. Use serum-free medium and complete growth medium that do not contain these or other polyanions.

Q17. Should I expect toxicity when using the TransIT®-mRNA Transfection Kit? How can I minimize cellular toxicity?
We observe little to no cellular toxicity using the TransIT®-mRNA Transfection Kit with multiple cell types. Ensure that you understand and are adhering to the recommended protocol to obtain optimal results. Even slight variations in the procedure can adversely affect transfection efficiency.

Rock culture vessel following addition of RNA/mRNA Boost Reagent/TransIT®-mRNA transfection complexes - Mix thoroughly to evenly distribute the complexes to all of the cells. We recommend rocking the dish back and forth and from side to side; do not swirl or rotate the dish.
Optimize the amount of TransIT®-mRNA Reagent, mRNA Boost Reagent, or RNA was used in the transfection - Titrate the amount of component(s) used in the transfection. Refer to the TransIT®-mRNA Transfection Kit protocol for recommended starting conditions.
Optimize cell density (% confluence) at the time of transfection - We recommend performing the transfection when cells are 60-90% confluent.
Transfect cells in complete growth media (containing serum) - Form complexes in serum-free media, and add complexes to cells growing in complete growth media (containing serum). Higher efficiency and viability are observed when cells are transfected in complete growth media.
Use cells of similar passage number - If the passage number of the cells is too high or too low they may be more sensitive to transfection reagents. Maintain a similar passage number between experiments to ensure reproducibility.

For specific questions or concerns, please contact our Technical Support Team at 888.530.0801 or techsupport@mirusbio.com.

Frequently Asked Questions:

TransIT®-mRNA Transfection Kit

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

TROUBLESHOOTING QUESTIONS AND ANSWERS

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

TROUBLESHOOTING QUESTIONS AND ANSWERS

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