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[Bio101] siRNA Transfection of Mouse Bone Marrow-derived Macrophages
[Bio101] 小鼠骨髓源巨噬细胞的siRNA转染   

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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(巨噬细胞), Transfection(转染), SiRNA(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 (unpublished)].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.

简介

短干扰RNA(siRNA)是一种双链RNA分子,通常为20-25bp长,参与RNA干扰现象。 siRNA转染在该方案中用于在BMM'phi'细胞中敲低靶基因表达。 在60-80%汇合下用细胞转染两天后,击倒效率可以达到90%。

关键字:巨噬细胞, 转染, siRNA

材料和试剂

  1. RPMI 1640培养基(RPMI)(Life Technologies,Invitrogen TM ,目录号:11875-093)
  2. 胎牛血清(Atlanta Biologicals,目录号:S10350)
  3. 青霉素/链霉素(P/S)(Life Technologies,Invitrogen TM,目录号:15140-122)
  4. Lipofectamine RNAiMAX(iMAX)(Life Technologies,Invitrogen TM ,目录号:13778150)

设备

  1. 单元格计数器
  2. 6孔板
  3. 24孔板

程序

  1. 小鼠骨髓来源的巨噬细胞(BMM'phi)的分离和培养(Chen,2011) 。
  2. 使用细胞计数器计数胰蛋白酶化的BMM'phi,然后将2×10 6个/ml细胞分入6孔板(用于实时PCR)或24孔板(用于分枝杆菌感染)中, 在BMM'phi'生长培养基中过夜
  3. Mix-1:RPMI500μl(6孔板)或100μl(24孔板)40nM siRNA。
    Mix-2:RPMI500μl(6孔板)或100μl(24孔板),4.8μliMAx(6孔板)或1.2μliMAx(24孔板)。 将Mix-2加入Mix-1,短暂涡旋和旋转沉淀; 在室温下孵育20分钟
  4. 将BMM'phi'培养基更换为RMPI 2ml(6孔板)或500μl(24孔板),加入转染。 混合到孔中,并在37℃孵育4小时
  5. 改变为BMM'phi'生长培养基,在37℃孵育2天。 现在细胞准备进行进一步的实验。

致谢

这项工作由杭州高新区5050项目,杭州市海外退学基金资助,浙江省ZJ1000项目资助。 该方案是在美国加利福尼亚州斯坦福大学遗传学系的Cohen实验室开发的[Chen等人(未公开)]。

参考文献

  1. Chen,R。(2011)。 小鼠骨髓来源的巨噬细胞(BMM'phi')的分离和培养生物 -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)。 使用Lipofectamine 2000试剂进行高级转染:原代神经元,siRNA和高通量应用。 方法 33(2):95-103。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用: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