My siRNA-mediated gene knockdown isn’t working well—any troubleshooting suggestions? I’ve checked transfection efficiency, but the silencing effect remains weak.
Improving Transfection Efficiency: First, optimize transfection conditions—such as cell density, amount of transfection reagent, and transfection duration—according to the manufacturer’s instructions. Additionally, you may try transfecting other cell types if possible. If the experimental model restricts you to a specific cell type, consider switching to a different transfection reagent or using an alternative transfection method, such as electroporation.
Use Serum-Free Medium During Transfection: Serum can interfere with the formation of complexes between the transfection reagent and siRNA, thereby reducing transfection efficiency.
Optimize siRNA Concentration: The optimal working concentration of siRNA varies depending on the cell type and research objectives. It is influenced by the characteristics of the siRNA, the cell line, and the chosen analytical methods. A recommended starting concentration is 50 nM, but it is advisable to test a range of concentrations to achieve the best transfection efficiency. However, excessively high siRNA concentrations may be toxic to cells.
Optimize Cell Growth Conditions: Poor cell health or suboptimal growth conditions can negatively affect transfection efficiency. Ensure that cells are healthy and in the appropriate growth phase before transfection.
Redesign siRNA if Necessary: If transfection efficiency is satisfactory and other factors have been ruled out, but gene silencing is still suboptimal, consider redesigning the siRNA sequences for improved knockdown efficiency.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
Dear Chloe Sun
In order to improve siRNA mediated gene silencing, I would like to present you our Lullaby transfection reagent. It is a lipid-based reagent highly efficient that presents a very low index of toxicity.
A wide range of short nucleic acid sequences can be used without any problem and co-transfections as well as sequential transfection are also possible.
■ Moreover, among the hundreths of publications metnioning its use (http://www.ozbiosciences.com/module/citationfinder/default), Lullaby transfection reagent was selected by the Cancer Research UK Beatson Institute for siRNA screening approach and as stated by Emma J. Shanks, "... By far, our preferred reagent is Lullaby from OZ Biosciences. We have used this reagent in over 20 cell lines and have found it essential in enabling siRNA screening in hard-to-transfect cell lines such as those derived from GEMM pancreatic tumors and AML suspension cell lines, with minimal toxicity"1.
→ If you need any information or if you want to try Lullaby, please do not hesitate to contact me via ResearchGate or directly at:
best regards,
Cedric
1 - http://www.ncbi.nlm.nih.gov/pubmed/24661213
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