Abstract
Short interfering RNAs (siRNA) are a type of double-stranded RNA molecule, typically 20-25 bp long, that are involved in the phenomenon of RNA interference. siRNA transfection is employed in this protocol to knockdown target gene expression in BMM’phi’ cells. Two days after transfection with cells at 60-80% confluence, the knockdown efficiency can reach 90%.
Keywords: Macrophage, Transfection, SiRNA
Materials and Reagents
Equipment
Procedure
Acknowledgments
This work was funded by 5050 project by Hangzhou Hi-Tech District, Funding for Oversea Returnee by Hangzhou City, ZJ1000 project by Zhejiang Province. This protocol was developed in the Cohen Lab, Department of Genetics, Stanford University, CA, USA [Chen et al. (unpublished)].
References
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The transfection can be done in 1-2 weeks after the isolation of the BMM cells.
Sorry, the iMAX dose for a well of a 6-well plate should be 4.8ul (1.2ul is for a well of a 24-well plate).The efficiency is high, can usually reach 70%-90% knockdown in two-three days. For some very highly-expressed targets, you may need to increase the siRNA concentration to 50nM-100nM and the iMAX dose to 7.5ul for a well of a 6-well plate or 1.5 ul for a well of a 24-well plate. Also, for some low-expressed targets, you can decrease the siRNA concentration to 20nM-30nM and the iMAX dose to to 3.6ul for a well of a 6-well plate or 0.9 ul for a well of a 24-well plate.
Step 3 has been corrected according to A1