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Frequently Asked Questions | Transfection

TransIT®-LT1 Transfection Reagent

TransIT®-LT1 Transfection Reagent is a broad spectrum, low toxicity transfection reagent that provides high efficiency plasmid DNA delivery in many mammalian cell types. TransIT®-LT1 is a low toxicity, serum-compatible transfection reagent that eliminates the need for any culture medium change. TransIT®-LT1 is suitable for both transient and stable transfection and can be used for many applications such as gene expression, viral production, shRNA expression and promoter analysis.

GENERAL QUESTIONS AND ANSWERS

Q1. What does TransIT®-LT1 stand for?
Q2. What is the composition of the TransIT®-LT1 Reagent?
Q3. What are the differences between Mirus broad spectrum DNA transfection reagents - TransIT®-LT1, TransIT®-2020 and TransIT-X2®?
Q4. Can TransIT®-LT1 transfect adherent and suspension cells?
Q5. Can TransIT®-LT1 be used for reverse transfection?
Q6. Is TransIT®-LT1 suitable for transfecting siRNA or miRNA?
Q7. Can I transfect proteins with TransIT®-LT1?

PROTOCOL QUESTIONS AND ANSWERS

Q8. How should TransIT®-LT1 be stored?
Q9. Which serum-free medium should I use for preparing TransIT®-LT1:DNA transfection complexes?
Q10. How much TransIT®-LT1 and DNA should I use for transfection?
Q11. Will antibiotics interfere with my transfection?
Q12. How long should I leave the TransIT®-LT1:DNA transfection complexes on my cells?
Q13. Are cell density (% confluence) and passage number important factors during transfection?
Q14. How can I assess transfection efficiency for my cell type?

TROUBLESHOOTING QUESTIONS

Q15. I observe a precipitate during transfection complex formation. What can I do to prevent this?
Q16. What can I do to further improve transfection efficiency of my target cell type when using the TransIT®-LT1 Reagent?
Q17. Should I be concerned about cellular toxicity when using TransIT®-LT1?

GENERAL QUESTIONS AND ANSWERS

Q1. What does TransIT®-LT1 stand for?
TransIT®-LT1 was the first lipopolyplex transfection reagent that provides both high efficiency and Low Toxicity (LT) DNA delivery to mammalian cells and therefore named so.

Q2. What is the composition of the TransIT®-LT1 Reagent?
TransIT®-LT1 is a broad spectrum non-liposomal formulation comprised of a lipid and protein/polyamine mixture.

Q3. What are the differences between Mirus broad spectrum DNA transfection reagents - TransIT®-LT1, TransIT®-2020 and TransIT-X2®?
TransIT®-LT1, TransIT®-2020 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.

FeaturesTransIT-X2® Dynamic Delivery SystemTransIT®-2020 Transfection ReagentTransIT®-LT1 Transfection Reagent
Nucleic Acid TransfectedDNA, siRNA, miRNA, pre-miRNADNA onlyDNA only
Low Cellular Toxicity*****
(Minimal Toxicity)
*****
(Minimal Toxicity)
*****
(Exceptionally Low Toxicity)
High knockdowncheckmarkNot suitable for this application‡Not suitable for this application‡
Co-transfection of DNA and siRNAcheckmarkNot suitable for this applicationNot suitable for this application
Animal Origin FreecheckmarkcheckmarkNo

‡ 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.

Q4. Can TransIT®-LT1 transfect adherent and suspension cells?
Yes. We have successfully transfected a wide variety of adherent cell types and suspension cells using TransIT®-LT1. Please refer to the TransIT®-LT1 user protocol (PDF) for starting cell density recommendations.

Q5. Can TransIT®-LT1 be used for reverse transfection?
Yes. TransIT®-LT1 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 (PDF)).

Q6. Is TransIT®-LT1 suitable for transfecting siRNA or miRNA?
No. TransIT®-LT1 does not effectively deliver siRNA or miRNA. For high efficiency siRNA delivery and knockdown of target gene expression, we recommend our two broad spectrum siRNA transfection reagents: TransIT-TKO® and TransIT-siQUEST®.

Q7. Can I transfect proteins with TransIT®-LT1?
Yes. While we have not specifically tested this application, our customers have reported successful transfection of proteins with TransIT®-LT1 transfection reagent.

PROTOCOL QUESTIONS AND ANSWERS

Q8. How should TransIT®-LT1 be stored?
Store the TransIT®-LT1 Reagent at 4°C. Before each use, warm to room temperature and vortex gently.

Q9. Which serum-free medium should I use for preparing TransIT®-LT1:DNA transfection complexes?
For best results, we recommend using Opti-MEM® I Reduced-Serum Medium for preparing TransIT®-LT1: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.

Q10. How much TransIT®-LT1 and DNA should I use for transfection?
Both the volume of TransIT®-LT1 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®-LT1 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 2-8µl per 1 µg DNA, depending on the cell type, passage number, cell density, and incubation time. For specific recommendations, see the user protocol (PDF).

Q11. 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).

Q12. How long should I leave the TransIT®-LT1:DNA transfection complexes on my cells?
There is no need to remove the TransIT®-LT1: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.

Q13. Are cell density (% confluence) and passage number important factors during transfection?
Yes. An optimal cell density of ≥80% confluence at the time of transfection, is essential for successful transfection using TransIT®-LT1 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.

Q14. 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®-LT1 Reagent to deliver a reporter plasmid encoding luciferase, beta-galactosidase or green fluorescent protein (GFP).

TROUBLESHOOTING QUESTIONS

Q15. 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®-LT1 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 (PDF). 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.

Q16. What can I do to further improve transfection efficiency of my target cell type when using the TransIT®-LT1 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 ≥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®-LT1 Reagent to DNA ratio - Determine the optimal TransIT®-LT1 Reagent:DNA ratio by titrating the reagent from 2-8 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 (PDF).

Q17. Should I be concerned about cellular toxicity when using TransIT®-LT1?
TransIT®-LT1 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 ≥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®-LT1:DNA ratio by using carrier DNA such as an empty cloning vector.
  • TransIT®-LT1 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 (PDF).

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?
Q2. What is the composition of the TransIT-X2® Dynamic Delivery System?
Q3. Is TransIT-X2® Dynamic Delivery System similar to Polyethyeleneimine (PEI)?
Q4. What are the differences between Mirus broad spectrum DNA transfection reagents - TransIT-X2®, TransIT®-2020 and TransIT®-LT1?
Q5. What are the differences between Mirus broad spectrum siRNA transfection reagents - TransIT-X2®, TransIT-TKO® and TransIT-siQUEST®?
Q6. Can TransIT-X2® transfect...adherent cells and suspension cells?...primary and stem cells?...neuronal cells?
Q7. Can TransIT-X2® be used for reverse transfection?
Q8. Is TransIT-X2® suitable for transfecting siRNA or miRNA?
Q9. Can TransIT-X2® be used for cotransfecting plasmid DNA and siRNA?
Q10. How many transfections can be performed per 1.5 ml vial of TransIT-X2®?

PROTOCOL QUESTIONS AND ANSWERS

Q11. How should TransIT-X2® be stored?
Q12. What is the shelf-life of TransIT-X2®?
Q13. Which serum-free medium should I use for preparing TransIT-X2® and nucleic acid (DNA and/or siRNA) transfection complexes?
Q14. How much TransIT-X2® and nucleic acid (DNA and/or siRNA) should I use for transfection?
Q15. Will antibiotics interfere with my transfection?
Q16. How long should I leave the TransIT-X2® transfection complexes on my cells?
Q17. Are cell density (%confluence) and passage number important factors during transfection?
Q18. How can I assess transfection efficiency for my cell type?

TROUBLESHOOTING QUESTIONS

Q19. I observe a precipitate during transfection complex formation. What can I do to prevent this?
Q20. What can I do to further improve transfection efficiency of my target cell type when using the TransIT-X2® System?
Q21. What can I do to further improve target gene knockdown when using the TransIT-X2® System?
Q22. Should I be concerned about cellular toxicity when using TransIT-X2®?

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.

FeaturesTransIT-X2® Dynamic Delivery SystemTransIT®-2020 Transfection ReagentTransIT®-LT1 Transfection Reagent
Nucleic Acid TransfectedDNA, siRNA, miRNA, pre-miRNADNA onlyDNA only
Low Cellular Toxicity*****
(Minimal Toxicity)
*****
(Minimal Toxicity)
*****
(Exceptionally Low Toxicity)
High knockdowncheckmarkNot suitable for this application‡Not suitable for this application‡
Co-transfection of DNA and siRNAcheckmarkNot suitable for this applicationNot suitable for this application
Animal Origin FreecheckmarkcheckmarkNo

‡ 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. (PDF)

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 (PDF).

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 (PDF).

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 (PDF). 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 (PDF) 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 (PDF).

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 (PDF).

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 (PDF).

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?
Q2. What are the differences between Mirus' broad spectrum DNA transfection reagents - TransIT®-2020, TransIT®-LT1 and TransIT-X2®?
Q3. Can TransIT®-2020 transfect...adherent cells and suspension cells?...primary and stem cells?..insect cells?
Q4. Can TransIT®-2020 be used for reverse transfection?
Q5. Is TransIT®-2020 suitable for transfecting siRNA or miRNA?

PROTOCOL QUESTIONS AND ANSWERS

Q6. How should TransIT®-2020 be stored?
Q7. Which serum-free medium should I use for preparing TransIT®-2020:DNA transfection complexes?
Q8. How much TransIT®-2020 and DNA should I use for transfection?
Q9. Will antibiotics interfere with my transfection?
Q10. How long should I leave the TransIT®-2020:DNA transfection complexes on my cells?
Q11. Are cell density (% confluence) and passage number important factors during transfection?
Q12. How can I assess transfection efficiency for my cell type?

TROUBLESHOOTING QUESTIONS

Q13. I observe a precipitate during transfection complex formation. What can I do to prevent this?
Q14. What can I do to further improve transfection efficiency of my target cell type when using the TransIT®-2020 Reagent?
Q15. Should I be concerned about cellular toxicity when using TransIT®-2020?

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.

FeaturesTransIT-X2® Dynamic Delivery SystemTransIT®-2020 Transfection ReagentTransIT®-LT1 Transfection Reagent
Nucleic Acid TransfectedDNA, siRNA, miRNA, pre-miRNADNA onlyDNA only
Low Cellular Toxicity*****
(Minimal Toxicity)
*****
(Minimal Toxicity)
*****
(Exceptionally Low Toxicity)
High knockdowncheckmarkNot suitable for this application‡Not suitable for this application‡
Co-transfection of DNA and siRNAcheckmarkNot suitable for this applicationNot suitable for this application
Animal Origin FreecheckmarkcheckmarkNo

‡ 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...

 

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 (PDF)).

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 (PDF).

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 (PDF) 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 (PDF). 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 (PDF).

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 (PDF).

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?
Q2. What cell types have been successfully transfected with the TransIT®-mRNA Transfection Kit?
Q3. The TransIT®-mRNA Transfection Kit contains two components, the TransIT®-mRNA Reagent and mRNA Boost Reagent. What is the formulation of each component?
Q4. What are the recommendations for RNA production for transfection?
Q5. Is it necessary to cap and polyadenylate the in vitro transcript before transfection?
Q6. What sizes of RNA can be delivered using the TransIT®-mRNA Transfection Kit?
Q7. Can the TransIT®-mRNA Transfection Kit be used to transfect suspension cells?
Q8. How should I store the TransIT®-mRNA Transfection Kit?
Q9. Where can I find references in which other researchers have used the TransIT®-mRNA Transfection Kit?
Q10. Can I use the TransIT®-mRNA Transfection Kit 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?
Q12. Do I have to perform a media change after transfection with the TransIT®-mRNA Transfection Kit?
Q13. Will antibiotics in the culture media interfere with transfection efficiency?
Q14. After transfection using the TransIT®-mRNA Transfection Kit, what is the optimal time to assay cells?
Q15. Can I cotransfect two RNA transcripts using TransIT®-mRNA Transfection Kit ?

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q16. How can I improve transfection efficiency using the TransIT®-mRNA Transfection Kit?
Q17. Should I expect toxicity when using the TransIT®-mRNA Transfection Kit? How can I minimize cellular toxicity?

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 (PDF) 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.

TransIT-PRO® Transfection Kit

TransIT-PRO® Transfection Kit consists of a DNA transfection reagent and boost combination specifically developed for mammalian protein production in suspension 293 and CHO derived cells. The PRO Boost Reagent is optional and enhances expression in certain media formulations. TransIT-PRO® Transfection Reagent and PRO Boost Reagent are comprised of animal-origin free components that are compatible with many chemically defined media formulations. Use of TransIT-PRO® Transfection Kit eliminates the need for a culture medium change post-transfection and is suitable for both transient and stable transfection.

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of TransIT-PRO® Transfection Kit?
Q2. What is the mechanism of action of TransIT-PRO® Transfection Kit?
Q3. Can TransIT-PRO® Transfection Kit be used to transfect other suspension CHO derived lines (e.g. CHO-DG44 cells etc.)?
Q4. How does TransIT-PRO® compare to the FreeStyle™ System from Life Technologies?
Q5. What is the effect on cellular viability following transfection with TransIT-PRO® Transfection Reagent?
Q6. What kind of protein yield (mg/L) can I expect using the TransIT-PRO® Transfection Kit?

PROTOCOL QUESTIONS AND ANSWERS

Q7. How should TransIT-PRO® Transfection Kit be stored?
Q8. Which serum-free medium should I use for preparing TransIT-PRO®: PRO Boost:DNA transfection complexes?
Q9. How much TransIT-PRO®, PRO Boost and DNA do I need to use per 1 liter of culture?
Q10. What is the largest culture volume that you have used for TransIT-PRO® transient transfections?
Q11. Will antibiotics interfere with my transfection?
Q12. Is a media change required post-transfection?

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q13. What can I do to further improve transfection efficiency of my target cell type when using the TransIT-PRO® Transfection Kit?
Q14. Can growth media formulation impact transfection performance when using the TransIT-PRO® Transfection Kit?
Q15. Can I add supplements to the transfection media?

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of TransIT-PRO® Transfection Kit?
TransIT-PRO® Transfection Kit consists of two animal origin-free components - a DNA transfection reagent called TransIT-PRO® and an optional PRO Boost Reagent. The TransIT-PRO® Reagent is a proprietary lipid-polymer mixture and the PRO Boost Reagent is an organic compound.

Q2. What is the mechanism of action of TransIT-PRO® Transfection Kit?
The TransIT-PRO® Reagent complexes with negatively charged DNA to form lipopolyplexes that associate with the negatively charged cell membrane via electrostatic interactions. These lipopolyplexes are thought to be taken into the cell via endocytosis. PRO Boost is a transfection booster identified during an empirical screening of Mirus compounds. However, the mechanism of the PRO Boost reagent is not well understood but appears to be medium-dependent. PRO Boost works best when added directly to the complex, suggesting a role in complex formation or protection of the complexes during the entry process.

Q3. Can TransIT-PRO® Transfection Kit be used to transfect other suspension CHO derived lines (e.g. CHO-DG44 cells etc.)?
At Mirus, we have only tested adaptations of the FreeStyle™ CHO-S cells from Life Technologies. We have received positive feedback from customers testing the TransIT-PRO® Transfection Kit on other CHO-S derived cells including the DG44 lines.

Q4. How does TransIT-PRO® compare to the FreeStyle™ System from Life Technologies?
We observe equivalent or higher protein yields using TransIT-PRO® versus FreeStyle™ Max Transfection Reagent to transfect FreeStyle™ CHO-S cells adapted to FreeStyle™ CHO Expression medium. Additionally, TransIT-PRO® exhibits broader media compatibility compared to the FreeStyle™ Max reagent. For detailed data, please see the TransIT-PRO® product webpage.

Q5. What is the effect on cellular viability following transfection with TransIT-PRO® Transfection Reagent?
Similar to all transfection methods, cells divide less rapidly following transfection with TransIT-PRO® compared to non-transfected cells. Initial comparison studies with leading competitors suggest that lower cell densities are obtained following transfection with TransIT-PRO®; however, protein titers are equal or higher. This reduction in cell mass, but not protein yield, might be beneficial to many researchers during downstream processing.

Q6. What kind of protein yield (mg/L) can I expect using the TransIT-PRO® Transfection Kit?
Protein yield is highly dependent on the intrinsic properties of the recombinant protein making it impossible to predict the protein yields of a particular construct. Furthermore, the nature of the suspension adapted cell types as well as the media formulation can have a huge impact on the final protein yield. Amongst antibody constructs that constitute a big fraction of biotherapeutic proteins, some subtypes (e.g. IgG1) express at higher levels. Ultimately, it is best to perform a side-by-side comparison with your current transfection method.

PROTOCOL QUESTIONS AND ANSWERS

Q7. How should TransIT-PRO® Transfection Kit be stored?
Both the TransIT-PRO® Transfection Reagent and PRO Boost Reagent should be stored at -20°C.

Q8. Which serum-free medium should I use for preparing TransIT-PRO®: PRO Boost:DNA transfection complexes?
For best results, we recommend using Opti-PRO™ Serum Free Medium (Opti-PRO™ SFM) for preparing transfection complexes using the TransIT-PRO® Transfection Kit. 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.

Q9. How much TransIT-PRO®, PRO Boost and DNA do I need to use per 1 liter of culture?
As a starting point, we recommend a starting TransIT-PRO® Reagent:PRO Boost:DNA ratio of 1:0.5:1 (1 ml of TransIT-PRO® reagent, 0.5 ml of PRO Boost Reagent and 1 mg of DNA per mL of culture). Therefore, 1 L of culture would require 1 mL of the TransIT-PRO® Transfection reagent and 0.5 mL of the optional PRO Boost Reagent.

Q10. What is the largest culture volume that you have used for TransIT-PRO® transient transfections?
Through collaboration, we have successfully transfected up to 4 liters of suspension CHO cells in a WAVE bioreactor. Additionally, we have received feedback from customers who have successfully transfected 1 liter bioreactors using the TransIT-PRO® Transfection Kit.

Q11. 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).

Q12. Is a media change required post-transfection?
No. A media change is not required following transfection with the TransIT-PRO® Transfection Kit. If desired, a media change can be performed at time points later than 4 hours post-transfection.

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q13. What can I do to further improve transfection efficiency of my target cell type when using the TransIT-PRO® Transfection Kit?
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 - Typically, a cell density of 0.5‒1.0 × 106 cells/ml at the time of transfection is desired. Ideally, cells should be split 18‒24 hours prior to transfection to ensure active cell division.
  • Suboptimal TransIT-PRO® Reagent to DNA ratio - Determine the optimal TransIT-PRO® Reagent:DNA ratio by titrating the reagent from 0.5-2 µl per 1 µg DNA.
  • Suboptimal PRO Boost to DNA ratio - Determine the optimal PRO Boost:DNA ratio by titrating the reagent from 0 -1.5 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.
  • Medium formulation not compatible with transfection - Some media do not exhibit good protein yields possibly due to components that inhibit transfection. For details on this topic, please refer to Q14-15.

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

Q14. Can growth media formulation impact transfection performance when using the TransIT-PRO® Transfection Kit?
Yes. Growth medium formulation can have a profound impact on transfection efficiencies. The TransIT-PRO® Transfection Kit displays broad media compatibility but certain media ingredients e.g. heparin, Pluronic F68, etc. may inhibit transfection. If your chosen media exhibits low protein yield, adaptation to compatible media may improve transfection performance.

Q15. Can I add supplements to the transfection media?
Yes. However, we recommend testing your transfection conditions with and without the media supplement to ensure that it does not inhibit transfection. Certain supplements such as 4mM L-glutamine does not impair transfection. If an inhibitory supplement such as Pluronic F68 is needed for long-term growth and increased protein yields, it can be added to the media 18-24 hours post-transfection.

TransIT-siQUEST® Transfection Reagent

TransIT-siQUEST® is a broad spectrum siRNA transfection reagent that enables high efficiency siRNA delivery and knockdown of target gene expression in many cell types including primary cells. Transfections with TransIT-siQUEST® do not require medium changes and can be carried out in serum-containing medium. In addition to TransIT-siQUEST®, Mirus also offers TransIT-TKO® Transfection Reagent for siRNA transfection. Each unique formulation provides high efficiency broad-spectrum siRNA delivery.

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of TransIT-siQUEST®?
Q2. Can TransIT-siQUEST® be used to transfect siRNA into primary cells?
Q3. Can TransIT-siQUEST® be used to transfect suspension cells?
Q4. Where can I find references in which other researchers have used TransIT-siQUEST®?
Q5. How does TransIT-siQUEST® Reagent differ from TransIT-TKO® Reagent?

PROTOCOL QUESTIONS AND ANSWERS

Q6. How should I store the TransIT-siQUEST® Reagent?
Q7. How should I dilute the siRNA?
Q8. Should I add the TransIT-siQUEST®/siRNA complexes to cells in serum-containing media or serum-free media?
Q9. Do I have to change the media or add media after transfection with TransIT-siQUEST®/siRNA?
Q10. Will antibiotics interfere with my transfection efficiency?
Q11. Can I co-transfect plasmid DNA and siRNA using TransIT-siQUEST®?
Q12. Can I transfect two different siRNA duplexes at the same time with TransIT-siQUEST® Reagent?
Q13. Can different sequence siRNA duplexes achieve different levels of knockdown of the same target gene?
Q14. How can I assess siRNA transfection efficiency for my cell type?

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q15. What can I do to improve knockdown efficiency when using the TransIT-siQUEST® Reagent?
Q16. Should I be concerned about toxicity when transfecting cells using TransIT-siQUEST®?

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of TransIT-siQUEST®?
TransIT-siQUEST® Transfection Reagent is a cationic proprietary polymer/lipid formulation, and is non-liposomal.

Q2. Can TransIT-siQUEST® be used to transfect siRNA into primary cells?
Yes. We have successfully transfected a number of primary cells including primary mouse hepatocytes. For other cell types, please refer to our Reagent Agent® Cell Transfection Database.

Q3. Can TransIT-siQUEST® be used to transfect suspension cells?
Yes. Generally, higher cell densities (2-4 fold higher than adherent cells) are recommended when transfecting suspension cells. Please refer to the TransIT-siQUEST® user protocol (PDF) for starting cell density recommendations.

Q4. Where can I find references in which other researchers have used TransIT-siQUEST®?
The most recent references reporting successful transfection using TransIT-siQUEST® can be found in our Citation Database.

Q5. How does TransIT-siQUEST® Reagent differ from TransIT-TKO® Reagent?
Due to their distinct formulations, each reagent has a unique transfection profile depending on the cell line being transfected. Generally, one particular reagent will be better suited for a particular cell line. TransIT-siQUEST® is formulated for siRNA delivery only whereas TransIT-TKO® can be used for co-transfecting plasmid DNA and siRNA.

PROTOCOL QUESTIONS AND ANSWERS

Q6. How should I store the TransIT-siQUEST® Reagent?
The TransIT-siQUEST® Reagent should be stored at 4°C and tightly capped to prevent evaporation.

Q7. How should I dilute the siRNA?
Use 100 mM NaCl in 50 mM Tris , pH 7.5, made with RNase-free water. Do not use water alone to dilute siRNA, as this may result in denaturation of the siRNA duplex, especially at low concentrations. siRNA can also be diluted in annealing buffer that is supplied with your siRNA.

Q8. Should I add the TransIT-siQUEST®/siRNA complexes to cells in serum-containing media or serum-free media?
Highest transfection efficiencies are achieved when the complexes are added to cells in their complete media (serum-containing). However, be sure to form the complexes in serum-free media before transfection, as serum can interfere with complex formation.

Q9. Do I have to change the media or add media after transfection with TransIT-siQUEST®/siRNA?
No. There is no need to change fresh culture medium after transfection. If required, perform a medium change at least 4 hours post-transfection to ensure adequate uptake of transfection complexes.

Q10. Will antibiotics interfere with my transfection efficiency?
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).

Q11. Can I co-transfect plasmid DNA and siRNA using TransIT-siQUEST®?
No. Due to the cationic nature of the TransIT-siQUEST® Reagent, it is recommended that plasmid DNA is delivered first using a DNA transfection reagent, followed by siRNA transfection using TransIT-siQUEST® Reagent 4 hours later. Refer to the TransIT-siQUEST® Reagent user protocol (PDF) for recommendations on performing this type of sequential transfection. If you want to simultaneously co-transfect plasmid DNA and siRNA, you can use our alternative siRNA transfection reagent TransIT-TKO®. For further details, please refer to the TransIT-TKO® user protocol (PDF).

Q12. Can I transfect two different siRNA duplexes at the same time with TransIT-siQUEST® Reagent?
Yes. The TransIT-siQUEST® Reagent can be used to transfect two different siRNA duplexes at the same time. No adjustment in the amount of TransIT-siQUEST® Reagent per well is needed; however, the total concentration of siRNAs should be maintained between 10-50 nM.

Q13. Can different sequence siRNA duplexes achieve different levels of knockdown of the same target gene?
Yes. We have found that different siRNA sequences may result in different levels of target gene knockdown. Try a few different sequences for a particular target gene if you are not achieving the knockdown efficiency that you expect.

Q14. How can I assess siRNA transfection efficiency for my cell type?
To assess siRNA delivery, the siRNA can be fluorescently labeled using Label IT® siRNA Tracker™ Intracellular Localization Kits and then visualized under a microscope. You can also indirectly measure transfection efficiency using functional assays such as western blots and RT-PCR for knockdown.

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q15. What can I do to improve knockdown efficiency when using the TransIT-siQUEST® Reagent?
Ensure that you understand and are adhering to the recommended protocol. Small variations in the procedure can affect transfection efficiency. Following are the most important factors that might affect your results:

  • Suboptimal volume of TransIT-siQUEST® Reagent - Titrate the reagent volume from 1-4 μl per well of a 24-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.

Q16. Should I be concerned about toxicity when transfecting cells using TransIT-siQUEST®?
No. If you are working with a sensitive cell type, the following factors might affect cell health

  • Excessive amount of TransIT-siQUEST® Reagent - Reduce the amount of TransIT-siQUEST® Reagent used for preparing complexes.
  • High siRNA concentration - Very low concentrations such as 1-5 nM have been used successfully to achieve knockdwon with TransIT-siQUEST®.
  • Low cell density - Grow cells to a higher cell density and repeat the transfection.
  • Media change or addition may be necessary in some cell lines - If incubating for 48-72 hours, it may be necessary to change the complete media 24 hours post-transfection.
  • Uneven distribution of TransIT-siQUEST® Reagent/siRNA complexes - Mix thoroughly and add complexes dropwise to all the areas of the wells. Rocking the dish back and forth and from side to side is recommended. Do not swirl or rotate the dish, as this may result in uneven distribution.
  • Include proper controls - Include the following controls to aid in assessing cellular toxicity: cells alone (for visual comparisons), TransIT- siQUEST Reagent alone, and TransIT-siQUEST® Reagent plus a non-specific siRNA.

 

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

TransIT-TKO® Transfection Reagent

TransIT-TKO® is a broad spectrum siRNA transfection reagent that enables high efficiency siRNA delivery and knockdown of target gene expression in many cell types including primary cells. TransIT-TKO® was introduced in 2001 and was the first commercially available siRNA transfection reagent. Co-transfection of siRNA and DNA is also feasible with TransIT-TKO® and has been tested across a variety of cell types. Transfections with TransIT-TKO® do not require medium changes and can be carried out in serum-containing medium. In addition to TransIT-TKO®, Mirus also offers TransIT-siQUEST® Transfection Reagent for siRNA transfection. Each unique formulation provides high efficiency broad-spectrum siRNA delivery.

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of TransIT-TKO®?
Q2. Can TransIT-TKO® be used to transfect siRNA into primary cells?
Q3. Can TransIT-TKO® be used to transfect suspension cells?
Q4. Where can I find references in which other researchers have used TransIT-TKO®?
Q5. How does TransIT-siQUEST® Reagent differ from TransIT-TKO® Reagent?

PROTOCOL QUESTIONS AND ANSWERS

Q6. How should I store the TransIT-TKO® Reagent?
Q7. How should I dilute the siRNA?
Q8. Should I add the TransIT-TKO®/siRNA complexes to cells in serum-containing media or serum-free media?
Q9. Do I have to change the media or add media after transfection with TransIT-TKO®/siRNA?
Q10. Will antibiotics interfere with my transfection efficiency?
Q11. Can I co-transfect plasmid DNA and siRNA using TransIT-TKO®?
Q12. Can I transfect two different siRNA duplexes at the same time with TransIT-TKO® Reagent?
Q13. Can different sequence siRNA duplexes achieve different levels of knockdown of the same target gene?
Q14. How can I assess siRNA transfection efficiency for my cell type?

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q15. What can I do to improve knockdown efficiency when using the TransIT-TKO® Reagent?
Q16. Should I be concerned about toxicity when transfecting cells using TransIT-TKO®?

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of TransIT-TKO®?
TransIT-TKO® Transfection Reagent is a cationic proprietary polymer/lipid formulation, and is non-liposomal.

Q2. Can TransIT-TKO® be used to transfect siRNA into primary cells?
Yes. We have successfully transfected a number of primary cells including human astrocytes, human neurons, and mouse hepatocytes. For other cell types, please refer to our Reagent Agent® Cell Transfection Database.

Q3. Can TransIT-TKO® be used to transfect suspension cells?
Yes. Generally, higher cell densities (2-4 fold higher than adherent cells) are recommended when transfecting suspension cells. Please refer to the TransIT-TKO® user protocol (PDF) for starting cell density recommendations.

Q4. Where can I find references in which other researchers have used TransIT-TKO®?
The most recent references reporting successful transfection using TransIT-TKO® can be found in our Citation Database.

Q5. How does TransIT-siQUEST® Reagent differ from TransIT-TKO® Reagent?
Due to their distinct formulations, each reagent has a unique transfection profile depending on the cell line being transfected. Generally, one particular reagent will be better suited for a particular cell line. TransIT-siQUEST® is formulated for siRNA delivery only whereas TransIT-TKO® can be used for co-transfecting plasmid DNA and siRNA.

PROTOCOL QUESTIONS AND ANSWERS

Q6. How should I store the TransIT-TKO® Reagent?
The TransIT-TKO® Reagent should be stored at 4°C and tightly capped to prevent evaporation.

Q7. How should I dilute the siRNA?
Use 100 mM NaCl in 50 mM Tris , pH 7.5, made with RNase-free water. Do not use water alone to dilute siRNA, as this may result in denaturation of the siRNA duplex, especially at low concentrations. siRNA can also be diluted in annealing buffer that is supplied with your siRNA.

Q8. Should I add the TransIT-TKO®/siRNA complexes to cells in serum-containing media or serum-free media?
Highest transfection efficiencies are achieved when the complexes are added to cells in their complete media (serum-containing). However, be sure to form the complexes in serum-free media before transfection, as serum can interfere with complex formation.

Q9. Do I have to change the media or add media after transfection with TransIT-TKO®/siRNA?
No. There is no need to change fresh culture medium after transfection. If required, perform a medium change at least 4 hours post-transfection to ensure adequate uptake of transfection complexes.

Q10. Will antibiotics interfere with my transfection efficiency?
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).

Q11. Can I co-transfect plasmid DNA and siRNA using TransIT-TKO®?
Yes, you can simultaneously co-transfect plasmid DNA and siRNA using TransIT-TKO®. Prepare DNA and siRNA transfection complexes in separate tubes and mix them together prior to adding them to the cells. For further details, please refer to the TransIT-TKO® user protocol (PDF).

Q12. Can I transfect two different siRNA duplexes at the same time with TransIT-TKO® Reagent?
Yes. The TransIT-TKO® Reagent can be used to transfect two different siRNA duplexes at the same time. No adjustment in the amount of TransIT-TKO® Reagent per well is needed; however, the total concentration of siRNAs should be maintained between 10-50 nM.

Q13. Can different sequence siRNA duplexes achieve different levels of knockdown of the same target gene?
Yes. We have found that different siRNA sequences may result in different levels of target gene knockdown. Try a few different sequences for a particular target gene if you are not achieving the knockdown efficiency that you expect.

Q14. How can I assess siRNA transfection efficiency for my cell type?
To assess siRNA delivery, the siRNA can be fluorescently labeled using Label IT® siRNA Tracker™ Intracellular Localization Kits and then visualized under a microscope. You can also indirectly measure transfection efficiency using functional assays such as western blots and RT-PCR for knockdown.

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q15. What can I do to improve knockdown efficiency when using the TransIT-TKO® Reagent?
Ensure that you understand and are adhering to the recommended protocol. Small variations in the procedure can affect transfection efficiency. Following are the most important factors that might affect your results:

  • Suboptimal volume of TransIT-TKO® Reagent - Titrate the reagent volume from 1-4 μl per well of a 24-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.

Q16. Should I be concerned about toxicity when transfecting cells using TransIT-TKO®?
No. If you are working with a sensitive cell type, the following factors might affect cell health

  • Excessive amount of TransIT-TKO® Reagent - Reduce the amount of TransIT-TKO® Reagent used for preparing complexes.
  • High siRNA concentration - Very low concentrations such as 1-5 nM have been used successfully to achieve knockdwon with TransIT-TKO®.
  • Low cell density - Grow cells to a higher cell density and repeat the transfection.
  • Media change or addition may be necessary in some cell lines - If incubating for 48-72 hours, it may be necessary to change the complete media 24 hours post-transfection.
  • Uneven distribution of TransIT-TKO® Reagent/siRNA complexes - Mix thoroughly and add complexes dropwise to all the areas of the wells. Rocking the dish back and forth and from side to side is recommended. Do not swirl or rotate the dish, as this may result in uneven distribution.
  • Include proper controls - Include the following controls to aid in assessing cellular toxicity: cells alone (for visual comparisons), TransIT-TKO® Reagent alone, and TransIT-TKO® Reagent plus a non-specific siRNA.

 

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

 


Technical Resources

Don’t See Your Cell Type? Consult Reagent Agent® Transfection Database
Citation Database: Check if our reagents have been used by other researchers to transfect your cell type
Technical Support: Communicate directly with a transfection expert

"We recently engineered a bispecific immunofusion for the treatment and elimination of leukemia stem cells. For this work we chose TransIT-PRO® for antibody production in CHO-S cells based on the high protein yield we obtained. (Kuo et al. Protein Eng Des Sel. 2012 Oct;25(10):561-9. Epub 2012 Jun 27)."
Jen-Sing Liu, Ph.D.
Molecular Templates Inc.