Frequently Asked Questions:

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 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 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.

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.

Questions?

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