Frequently Asked Questions:

TransIT-X2® Dynamic Delivery System

TransIT-X2® Dynamic Delivery System is an advanced, non-liposomal polymeric system that enables high-efficiency transfection of many cell types, including primary cells. TransIT-X2® can be used for DNA delivery, siRNA/miRNA delivery or simultaneous delivery of both DNA and siRNA. This reagent is suitable for both transient and stable transfection. TransIT-X2® 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 does TransIT-X2® mean?
TransIT® is the trusted brand of transfection reagents offered by Mirus ever since 1995; there are over 1600 published citations using Mirus TransIT® in vitro transfection reagents, on atleast 492 unique cell lines and primary cells (see the Citations database). Mirus expanded its offering with the latest TransIT-X2® Dynamic Delivery system that gives researchers several advantages over other transfection reagents, as follows:

icon Efficiency-exceptional broad spectrum transfection
icon Versatility-cutting edge delivery of plasmid DNA and siRNA
icon Technology-novel non-liposomal, polymeric delivery

Q2. What is the composition of the TransIT-X2® Dynamic Delivery System?
TransIT-X2® Dynamic Delivery System is an advanced non-liposomal system that comprises of a completely novel class of polymers in addition to other proprietary components that aid in nucleic acid complexation, uptake and endosomal release thereby enabling superior transfection of both plasmid DNA and smaller nucleic acids such as siRNA/miRNA/pre-miRNA. TransIT-X2® also enables the co-delivery plasmid DNA and siRNA without compromising transfection efficiency. TransIT-X2® does not contain any components of animal origin.

Q3. Is TransIT-X2® Dynamic Delivery System similar to Polyethyeleneimine (PEI)?
No. TransIT-X2® is a completely novel polymeric formulation and is not Polyethyleneimine (PEI)- based.

Q4. What are the differences between Mirus broad spectrum DNA transfection reagents - TransIT-X2®, TransIT®-2020 and TransIT®-LT1?
TransIT-X2®, TransIT®-2020 and TransIT®-LT1 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.

Q5. What are the differences between Mirus broad spectrum siRNA transfection reagents - TransIT-X2®, TransIT-TKO® and TransIT-siQUEST®?
TransIT-X2®, TransIT-TKO® and TransIT-siQUEST® are all high-efficiency broad spectrum formulations for siRNA delivery that are chemically distinct from each other. 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 co-transfecting plasmid DNA and siRNA. TransIT-TKO® and TransIT-siQUEST® are suitable for effective gene knockdown. Depending on the cell type, one reagent may have superior performance over the others. For cell-type specific recommendations, please consult the Reagent Agent® transfection database.

Q6. Can TransIT-X2® transfect...

Adherent and suspension cells? Yes. We have successfully transfected a wide variety of adherent cell types (e.g., CHO-K1, Hep G2, primary human mammary epithelial cells, etc.) and suspension cells such as Freestyle 293-F cells using TransIT-X2®. Please refer to the TransIT-X2® user protocol for starting cell density recommendations.
Primary cells? Yes. TransIT-X2® can be used to efficiently transfect primary cells. At Mirus, we have successfully transfected HUVECs, primary human mammary epithelial cells (HMEC) and normal human dermal fibroblasts (NHDF) with high efficiency.
Neuronal cells? Yes. We have successfully transfected the following neuronal cell types/models in our labs: iCell® Neurons, SH-SY5Y, SK-N-MC and PC-12.

View a complete listing of cell types transfected using TransIT-X2® by Mirus scientists and corresponding starting reagent-to-nucleic acid ratios.

Q7. Can TransIT-X2® be used for reverse transfection?
Yes. TransIT-X2® 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. For additional technical and protocol considerations, please refer to Tips from the Bench - Forward and Reverse Transfection Protocols. For a detailed plasmid DNA reverse transfection protocol, view Tips from the Bench - Reverse Transfection Protocol for Plasmid DNA. For a detailed siRNA/miRNA reverse transfection protocol, view Tips from the Bench - Reverse Transfection Protocol for siRNA/miRNA.

Q8. Is TransIT-X2® suitable for transfecting siRNA or miRNA?
Yes. TransIT-X2® efficiently delivers smaller nucleic acids such as siRNA/miRNA and pre-miRNA into mammalian cells. Detailed instructions for siRNA delivery can be found in the user protocol.

Q9. Can TransIT-X2® be used for cotransfecting plasmid DNA and siRNA?
TransIT-X2® can be used for simultaneous delivery of DNA and siRNA. A detailed DNA and siRNA cotransfection protocol can be found in the user protocol.

Q10. How many transfections can be performed per 1.5 ml vial of TransIT-X2®?
The number of transfections per 1.5 ml can be variable as the optimal TransIT-X2®: nucleic acid ratio is dependent on several factors including cell type, plate format, etc. As an example, for a cell type that transfects optimally at 3:1 TransIT-X2®: nucleic acid ratio (i.e. 3 µl TransIT-X2® per 1 µg DNA), approximately 500 wells of a 12 -well plate can be transfected using a 1.5 ml vial (MIR 6000). View TransIT-X2® product configurations.

PROTOCOL QUESTIONS AND ANSWERS

Q11. How should TransIT-X2® be stored?
Store the TransIT-X2® Reagent at -20°C.

Q12. What is the shelf-life of TransIT-X2®?
Mirus guarantees this product for 1 year from date of purchase when properly stored and handled.

Q13. Which serum-free medium should I use for preparing TransIT-X2® and nucleic acid (DNA and/or siRNA) transfection complexes?
For best results, we recommend using Opti-MEM® I Reduced-Serum Medium for preparing TransIT-X2®:nucleic acid (DNA and/or siRNA) 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.

Q14. How much TransIT-X2® and nucleic acid (DNA and/or siRNA) should I use for transfection?
Both the volume of TransIT-X2® and amount of nucleic acid needed for transfection is dependent on the surface area of the tissue culture well/vessel. We have the following recommendations depending on the type of nucleic acid being transfected.

DNA transfection: Our starting recommendation is 7.5 µl TransIT-X2® and 2.5 µg DNA which represents a 3:1 ratio (µl of reagent/µg of DNA) per well of a 6-well plate. For best results, we recommend further optimization of this reagent to DNA ratio from 2-6 µl per 1 µg DNA, depending on the cell type, passage number, cell density, and incubation time.
siRNA/miRNA/pre-miRNA transfection: As a starting point, test 7.5 µl of TransIT-X2® per well of a 6-well plate when transfecting small RNA at a final concentration of 25 nM. For further optimization, test three amounts of TransIT-X2®, e.g. 5 µl, 7.5 µl, and 10 µl per well of a 6-well plate.
DNA and siRNA co-transfection: Our starting recommendation is 7.5 µl TransIT-X2®, 2.5 µg DNA and 25 nM siRNA per well of a 6-well plate. Vary the amount of TransIT-X2® from 2-6 µl per 1 µg DNA to find the optimal ratio.

NOTE: For specific recommendations on the above, see the user protocol. Optimal starting conditions for many cell types can be found using our online Reagent Agent® transfection database.

Q15. 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 to 0.1-1X final concentration).

Q16. How long should I leave the TransIT-X2®: nucleic acid transfection complexes on my cells?
There is no need to remove the TransIT-X2®: nucleic acid 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.

Q17. Are cell density (%confluence) and passage number important factors during transfection?
Yes. An optimal cell density, ≥ 80% confluence at the time of transfection, is essential for successful transfection using TransIT-X2® 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.

Q18. How can I assess transfection efficiency for my cell type?
To verify gene expression post-transfection, use TransIT-X2® System to deliver a reporter plasmid encoding luciferase, beta-galactosidase or green fluorescent protein (GFP). For details, please refer to our Tips from the Bench - Measure Transfection Efficiency. To specifically 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. For details, please refer to our Tips from the Bench - Track Intracellular Nucleic Acid Delivery. Likewise, to assess delivery efficiency of siRNA, use Mirus’ Label IT® siRNA Tracker™ Intracellular Localization Kits or prelabeled Label IT® RNAi Delivery Controls.

TROUBLESHOOTING QUESTIONS

Q19. I observe a precipitate during transfection complex formation. What can I do to prevent this?
Precipitation may be observed when excess plasmid DNA is used during complex formation. This may negatively impact transfection efficiency. As a general practice, scale all reagents including serum-free medium, TransIT-X2® and 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.

Q20. What can I do to further improve transfection efficiency of my target cell type when using the TransIT-X2® System?
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 ≥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-X2® Reagent to DNA ratio - Determine the optimal TransIT-X2®:DNA ratio by titrating the reagent from 2-6 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.

Q21. What can I do to further improve target gene knockdown when using the TransIT-X2® System?
Small deviations from the procedure outlined in the TransIT-X2® protocol can dramatically impact knockdown efficiency. Following are the most important factors that might affect your results:

Suboptimal volume of TransIT-X2® - Titrate the reagent volume from 5-10 μl per well of a 6-well plate.
Suboptimal siRNA concentration - Vary the siRNA concentration between 10-50 nM final concentration in the well.
Denatured siRNA - Use recommended buffer (100 mM NaCl, 50 mM Tris, pH 7.5 in RNase-free water) or annealing buffer to dilute siRNA. Do not use water as this can denature the siRNA duplex.
Poor quality of transfecting siRNA - Avoid siRNA degradation by using RNase-free handling procedures and plasticware. Degradation can be detected on acrylamide gels.
Incorrect siRNA sequence - Ensure that the sequence of siRNA is correct for your gene of interest.
Off-target effects - Transfect a non-targeting or nonsense siRNA control sequence to verify that the gene expression knockdown or phenotype is attributed to the genespecific siRNA. Additionally, targeting a gene with multiple siRNA sequences ensures that the resulting phenotype is not due to off-target effects.
Inhibitor present during transfection - The presence of polyanions, such as dextran sulfate or heparin, can inhibit transfection. Use transfection medium that does not contain these polyanions.
Poor detection of gene knockdown - The target mRNA is usually degraded within the first 24 hours post-transfection and can be measured using assays such as qRT-PCR and Northern blots. When using protein-based assays (Western blots, ELISA’s, etc), the stability of the target protein should be taken into consideration when determining the optimal time to assay the cells after transfection.

Q22. Should I be concerned about cellular toxicity when using TransIT-X2®?
TransIT-X2® is a low-toxicity formulation 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 ≥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-X2®:DNA ratio by using carrier DNA such as an empty cloning vector.
siRNA knockdown of an essential gene -If you are transfecting siRNA that is directed against a gene that is essential to the cell, cytotoxicity may be observed due to knockdown of the target gene. Include a transfection control with non-targeting siRNA to compare the cytotoxic effects of the gene being knocked down.
TransIT-X2® 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.
Medium change or addition - If incubating cells for long durations such as 48-72 hours post transfection, it may be necessary to replace the complete medium 24 hours post-transfection.

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

Frequently Asked Questions:

TransIT-X2® Dynamic Delivery System

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

TROUBLESHOOTING QUESTIONS

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

TROUBLESHOOTING QUESTIONS

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