Optimize the siIMPORTER™/siRNA ratio by varying the amount of siIMPORTER™, while keeping a constant amount of siRNA.
Suboptimal siRNA concentration
After establishing the optimal siIMPORTER™/siRNA ratio, vary the siRNA quantity over the ranges suggested in the Protocol section.
Poor siRNA quality
Use RNase-free handling procedures and plastic ware. Check for siRNA degradation on acrylamide gels.
Denatured siRNA
Use recommended buffer (cat. #20-272) to dilute siRNA. Water is not recommended as it can denature siRNA.
Cells have been in continuous passage for > 2 months
Thaw and culture fresh vial of cells that are from a low passage number. Avoid using high passage number cells.
Suboptimal cell density
Use cells that are 40-60% confluent on the day of transfection. Optimal cell density may vary depending on cell type.
Improper storage
siIMPORTER™ is very stable but long exposure to elevated temperatures and/or freeze/thaw cycles may cause degradation of the reagent. Store siIMPORTER™ at 4?C.
Wrong medium
Be sure to use serum-free medium when the siIMPORTER™ complex.
Serum in media on cells
Use serum-free medium during transfection and boost with serum-containing media 4 hours later.
Adverse siRNA diluent effect
In some cell types, the diluent causes decreased transfection efficiencies.
Cell line is difficult to transfect
Optimize siIMPORTER™/siRNA ratio and siRNA amount as indicated in the Optimization section VI.
siIMPORTER™/siRNA complexes were not freshly prepared
Prepare siIMPORTER™/siRNA complexes just before use and store them no longer than 30 minutes.
Suboptimal siIMPORTER™/siRNA ratio used
Too much siIMPORTER™ or too much siRNA could cause aggregation. Adjust the ratio as outlined above.
Aggregation
Excess siIMPORTER™ used
Decrease amount of siIMPORTER™ used.
Cytotoxicity
Unhealthy cells
1. Check cells for contamination.
2. Thaw a new batch of cells.
3. Cells are too confluent or cell density too low.
4. Check culture medium (pH, type used, last time checked, etc.)
5. Check materials used for proper function (culture plates, incubator temperatures, etc.)
siIMPORTER™ concentration too high
1. Reduce siIMPORTER™ concentration in 20-30% increments.
