Frequently Asked Questions:

TransIT®-2020 Transfection Reagent

TransIT®-2020 Transfection Reagent is a high-performance, low-toxicity, broad spectrum reagent that provides superior transfection of plasmid DNA into mammalian cells. TransIT®-2020 provides high levels of gene expression in hard-to-transfect cell types including primary and stem cells. This reagent is suitable for both transient and stable transfection. TransIT®-2020 is composed of animal-origin free components and is serum compatible, eliminating the need for any culture medium change after transfection.

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of the TransIT®-2020 Reagent?
TransIT®-2020 is a proprietary lipid-polymer mixture that forms lipopolyplexes with DNA. TransIT®-2020 does not contain any components of animal origin.

Q2. What are the differences between Mirus' broad spectrum DNA transfection reagents - TransIT®-2020, TransIT®-LT1 and TransIT-X2®?
TransIT®-2020, TransIT®-LT1 and TransIT-X2® are all high-efficiency broad spectrum formulations but are chemically distinct. Please see the table below for distinguishing features of each formulation. TransIT-X2® is our preferred recommendation for both superior gene expression as well as high knockdown. TransIT-X2® delivers both plasmid DNA and/or siRNA efficiently and can be used for cotransfecting plasmid DNA and siRNA. TransIT®-2020 and TransIT®-LT1 are suitable for plasmid DNA transfections only. TransIT-X2® and TransIT®-2020 are animal-origin free and are therefore should be the reagents of choice in a restrictive environment such as pharmaceutical development. Depending on the cell type, one reagent may have superior performance over others; for cell-type specific recommendations, please consult the Reagent Agent® transfection database.

Features	TransIT-X2® Dynamic Delivery System	TransIT®-2020 Transfection Reagent	TransIT®-LT1 Transfection Reagent
Nucleic Acid Transfected	DNA, siRNA, miRNA, pre-miRNA	DNA only	DNA only
Low Cellular Toxicity	***** (Minimal Toxicity)	***** (Minimal Toxicity)	***** (Exceptionally Low Toxicity)
High knockdown		Not suitable for this application‡	Not suitable for this application‡
Co-transfection of DNA and siRNA		Not suitable for this application	Not suitable for this application
Animal Origin Free			No

‡ Target gene knockdown can be accomplished with this reagent via shRNA encoding plasmid DNA delivery. This reagent is not optimal for the delivery of smaller nucleic acids such as siRNA/miRNA.

Q3. Can TransIT®-2020 transfect...

adherent and suspension cells? Yes. We have successfully transfected a wide variety of adherent cell types (e.g., HepG2, MCF-7, Neuro-2a, etc.) and suspension cells (e.g. K562, Jurkat, etc.) using TransIT®-2020. Please refer to the TransIT®-2020 user protocol for starting cell density recommendations. View a complete listing of cell lines transfected using TransIT®-2020 by Mirus scientists and other researchers.
primary and stem cells? Yes. TransIT®-2020 can be used to efficiently transfect primary and stem cells. View a complete listing of primary and stem cells transfected using TransIT®-2020 by Mirus scientists and other researchers.
insect cells? Yes. However, our primary recommendation for transfecting insect cells is TransIT®-Insect Transfection Reagent. Please refer to Reagent Agent® transfection database for insect cell line specific recommendations.

Q4. Can TransIT®-2020 be used for reverse transfection?
Yes. TransIT®-2020 can be used for reverse transfection as required for high-throughput screening projects. Transfection complexes may be formed in multiwell plates just prior to adding trypsinized cells. The cell density should be twice the recommended cell density (as per the user protocol).

Q5. Is TransIT®-2020 suitable for transfecting siRNA or miRNA?
No. TransIT®-2020 does not effectively deliver siRNA or miRNA. For high efficiency siRNA delivery and knockdown of target gene expression, TransIT-X2® is our preferred recommendation. TransIT-X2® delivers both plasmid DNA and/or siRNA efficiently and can be used for cotransfecting plasmid DNA and siRNA. Additional recommendations are our two broad spectrum siRNA transfection reagents: TransIT-TKO® and TransIT-siQUEST®; for cell-type specific recommendations, please consult the Reagent Agent® transfection database.

PROTOCOL QUESTIONS AND ANSWERS

Q6. How should TransIT®-2020 be stored?
Store the TransIT®-2020 Reagent at -20°C.

Q7. Which serum-free medium should I use for preparing TransIT®-2020:DNA transfection complexes?
For best results, we recommend using Opti-MEM® I Reduced-Serum Medium for preparing TransIT®-2020:DNA transfection complexes. Some serum free media contain polyanions such as heparin, or polydextran sulfate that may inhibit formation of active transfection complexes. If it is necessary to use media containing polyanions for your experimental set-up, the transfection medium can be replaced with polyanion containing medium 24 hours post transfection.

Q8. How much TransIT®-2020 and DNA should I use for transfection?
Both the volume of TransIT®-2020 and amount of DNA needed for transfection is dependent on the surface area of the tissue culture well/vessel. Per well of a 6-well plate, our starting recommendation is 7.5 µg TransIT®-2020 and 2.5 µg DNA which represents a 3:1 ratio (µl of reagent/µg of DNA). For best results, we recommend further optimization of this reagent to DNA ratio from 1-4 µl per 1 µg DNA, depending on the cell type, passage number, cell density, and incubation time. For specific recommendations, see the user protocol.

Q9. Will antibiotics interfere with my transfection?
Some antibiotics such as Kanamycin are cationic and will inhibit transfection complex formation. We recommend excluding antibiotics from the complex formation step. However, once the transfection complexes are formed, they can be added to cells grown in complete culture medium containing low levels of antibiotics (e.g.100X stock of penicillin/streptomycin diluted up to 0.1–1X final concentration).

Q10. How long should I leave the TransIT®-2020:DNA transfection complexes on my cells?
There is no need to remove the TransIT®-2020:DNA transfection complexes.If media change is required, we recommend leaving the transfection complexes on the cells for at least 4 hours before performing any medium change to ensure adequate uptake of the complexes.

Q11. Are cell density (% confluence) and passage number important factors during transfection?
Yes. An optimal cell density, 40-80% confluence at the time of transfection, is essential for successful transfection using TransIT®-2020 in most cell types. Please refer to the user protocol for specific cell density recommendations. Monitoring passage number is important since high passage number can alter cell characteristics such as morphology and division rate.

Q12. How can I assess transfection efficiency for my cell type?
To assess efficiency of plasmid DNA delivery, use Mirus’ Label IT® Tracker™ Nucleic Acid Intracellular Localization Kit to label target plasmid or choose Mirus’ prelabeled Label IT® Plasmid Delivery Controls. Alternatively, to verify gene expression post-transfection, use TransIT®-2020 Reagent to deliver a reporter plasmid encoding luciferase, beta-galactosidase or green fluorescent protein (GFP). For details, please refer to our Tips from the Bench - Track Intracellular Nucleic Acid Delivery and Measure Transfection Efficiency.

TROUBLESHOOTING QUESTIONS

Q13. I observe a precipitate during transfection complex formation. What can I do to prevent this?
Precipitation may be observed when excess DNA is used during complex formation. This may negatively impact transfection efficiency. As a general practice, scale all reagents including serum-free medium, TransIT®-2020 and plasmid DNA in linear proportion to the tissue culture vessel volume during complex formation. If precipitation is still observed due to high concentrations of DNA, increase the volume of serum-free medium during complex formation by two-fold. For details, please refer to the user protocol. Transfection complexes, visualized as small particles, are sometimes observed following transfection. These complexes are not toxic to cells and do not impact transfection efficiency or transgene expression.

Q14. What can I do to further improve transfection efficiency of my target cell type when using the TransIT®-2020 Reagent?
Multiple factors could contribute to low transfection efficiency; refer to the following critical troubleshooting tips to improve your transfection results:

Cell density (% confluence) not optimal at time of transfection - The recommended cell density for most cell types at the time of transfection is 40-80% confluence. However, it may be necessary to determine the best cell density for each cell type in order to maximize transfection efficiency.
Suboptimal TransIT®-2020 Reagent to DNA ratio - Determine the optimal TransIT®-2020 Reagent:DNA ratio by titrating the reagent from 1-4 µl per 1 µg DNA.
Poor quality of transfecting DNA - DNA used for transfection should be highly purified, sterile, and free from contaminants such as endotoxin. Remove any traces of endotoxin (bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit.
Complexes added to cells in serum-free medium - Form complexes in serum-free medium, and add to cells in complete (serum-containing) medium.

For additional tips, please refer to the Troubleshooting Guide in the user protocol.

Q15. Should I be concerned about cellular toxicity when using TransIT®-2020?
TransIT®-2020 is a low-toxicity transfection reagent that does not require any medium change after transfection. However, cellular toxicity might be observed with sensitive cell types or if the transfection protocol is not followed properly. Following are some suggestions to improve cell health post-transfection:

Cell density (% confluence) too low at time of transfection - The recommended cell density for most cell types at the time of transfection is 40-80% confluence. If cytotoxicity is observed, then increase the cell density to maintain cell health.
Plasmid DNA contains high levels of endotoxin - DNA used for transfection should be highly purified, sterile, and free from contaminants such as endotoxin that can cause cellular toxicity. Remove any traces of endotoxin (bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit.
Expressed target gene is toxic to cells - Compare toxicity levels against a cells alone control and cells transfected with an empty vector to assess the cytotoxic effects of the target protein being expressed. If lower levels of target gene expression are desired in your transfection experiments, consider reducing the amount of target plasmid. Maintain the optimal TransIT®-2020:DNA ratio by using carrier DNA such as an empty cloning vector.
TransIT®-2020 Reagent:DNA complexes were not mixed thoroughly with the cells in the well - Mix thoroughly to evenly distribute the complexes to all cells. Rock the dish back and forth and from side to side to properly distribute the complexes. Do not swirl or rotate the dish, as this may result in uneven distribution.
Complexes were added to the cells in serum-free medium - Form complexes in serum-free medium, and add to cells in complete (serum-containing) medium.
Cell morphology has changed - If the passage number of the cells is too high or too low, they can be more sensitive to transfection reagents. Maintain a similar passage number between experiments to ensure reproducibility.
Mycoplasma contamination affecting cell health - Check your cells for mycoplasma contamination. Use a fresh frozen stock of cells or use appropriate antibiotics to eliminate mycoplasma.

For additional tips, please refer to the Troubleshooting Guide in the user protocol.

Frequently Asked Questions:

TransIT®-2020 Transfection Reagent

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

TROUBLESHOOTING QUESTIONS

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

TROUBLESHOOTING QUESTIONS

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