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siRNA Transfection of Mouse Bone Marrow-derived Macrophages   

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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

  1. RPMI 1640 medium (RPMI) (Life Technologies, InvitrogenTM, catalog number: 11875-093 )
  2. Fetal bovine serum (Atlanta Biologicals, catalog number: S10350 )
  3. Stock penicillin/streptomycin (P/S) (Life Technologies, InvitrogenTM, catalog number: 15140-122 )
  4. Lipofectamine RNAiMAX (iMAX) (Life Technologies, InvitrogenTM, catalog number: 13778150 )

Equipments

  1. Cell counter
  2. 6-well plate
  3. 24-well plate

Procedure

  1. Isolation and culture of mouse bone marrow-derived macrophages (BMM’phi’) (Chen, 2011).
  2. Use cell counter to count trypsinized BMM’phi’s, and then split 2 x 106/ml cells into 6-well plates (for real-time PCR) or 24-well plates (for mycobacterial infection), in BMM’phi’ growth medium overnight.
  3. Mix-1: RPMI 500 μl (6-well plate) or 100 ul (24 well plate) 40 nM siRNA.
    Mix-2: RPMI 500 μl (6-well plate) or 100 μl (24 well plate), 4.8 μl iMAx (6-well plate) or 1.2 μl iMAx (24-well plate). Add Mix-2 to Mix-1, briefly vortex and spin-down; incubate for 20 min at room temperature.
  4. Change BMM’phi’ culture medium to RMPI 2 ml (6-well plate) or 500 μl (24- well plate), add the transfection. Mix to the well and incubate at 37 °C for 4 h.
  5. Change to BMM’phi’ growth medium, incubate at 37 °C for 2 days. Now the cells are ready for further experiments.

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 (unpublished)].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

  1. Chen, R. (2011). Isolation and culture of mouse bone marrow-derived macrophages (BMM’phi’). Bio-protocol 1(9): e68.
  2. Dalby, B., Cates, S., Harris, A., Ohki, E. C., Tilkins, M. L., Price, P. J. and Ciccarone, V. C. (2004). Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications. Methods 33(2): 95-103.
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Chen, R. (2011). siRNA Transfection of Mouse Bone Marrow-derived Macrophages. Bio-protocol Bio101: e28. DOI: 10.21769/BioProtoc.28.
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chunxiao hu
pharmocology of UIC
On which day should the transfection be done after the isolation of BMM?
9/30/2013 12:08:32 PM Reply
Ran Chen
Stanford University

The transfection can be done in 1-2 weeks after the isolation of the BMM cells.

10/2/2013 12:15:12 PM


Yuanqing Lin
what is the efficiency of transfection?
5/3/2011 6:28:54 AM Reply
bio-protocol

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.

5/4/2011 2:22:37 AM


bio-protocol

Step 3 has been corrected according to A1

5/4/2011 2:58:48 PM