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Dharmacon siRNA Transfection of HeLa Cells
Dharmacon siRNA 转染HeLa细胞   

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Abstract

Small Interfering RNA (siRNA) is a class of double-stranded RNAs of 20-25 nucleotides that play important roles in many biological processes (Hamilton and Baulcombe, 1999). siRNAs act by “neutralizing” the mRNA of the target protein, facilitating degradation of the mRNA and hence altering the biological effect of the protein (reviewed in Hannon and Rossi, 2004). siRNAs may also change the intracellular levels of regulatory RNAs. Use of siRNAs for manipulating the expression of genes of interest in biological research is commonly referred to as RNA interference or knockdown technique (Elbashir et al., 2001). Synthetic siRNAs are an emerging tool that are now widely used in these studies. A variety of algorithms are employed by different companies for the design of siRNA products, which differ in efficacy, specificity and cost among other criteria. An example protocol of siRNA knockdown is explained here using the siGENOME SMARTpool reagents from Dharmacon.

Materials and Reagents

  1. Human cervix epithelial carcinoma cell line Hela (ATCC, catalog number: CCL-2 ™)
  2. Eagle's Minimum Essential Medium (ATCC, catalog number: 30-2003 ™)
  3. Fetal bovine serum (FBS) (ATCC, catalog number: 30-2020 ™)
  4. 5x siRNA buffer (GE Healthcare Dharmacon, catalog number: B-002000-UB-100 )
  5. DharmaFECT 1 siRNA Transfection Reagent (GE Healthcare Dharmacon, catalog number: T-2001-01 )
  6. siGENOME SMARTpool reagents (GE Healthcare Dharmacon)
  7. Glyceral-dehyde-3-phosphate dehydrogenase (GAPD) (GE Healthcare Dharmacon, catalog number: D-001140-01 )
  8. Non-targeting siRNA control pool (GE Healthcare Dharmacon, catalog number: D-001206-13 )

Equipment

  1. 12-well polystyrene tissue culture plate (BD Biosciences, Falcon®, catalog number: 353043 )
  2. Cell culture incubator: 37 ºC and 5% CO2

Procedure

  1. Carry Hela cells in Eagle's Minimum Essential Medium with 10% FBS.
  2. Trypsinize, count cells and reseed cells 12-16 h before knockdown (see Note 1).
  3. Resuspend siRNA in 1x siRNA buffer to reach a final concentration of 5 μM.
  4. Add 5 μl of the 5 μM siRNA to 95 μl of serum-free medium in a low-adhesion tube 1, mix by gently tapping the tube or pipetting up and down.
  5. Add 0.5~5 μl DharmaFECT 1 reagent (see Note 2) to 99 μl of serum-free medium in a separate tube 2, mix by gently tapping the tube or pipetting up and down (see Note 3).
  6. The two tubes in steps 4-5 are incubated at room temperature for 5 min.
  7. Add the content of tube 1 from step 4 to tube 2 from step 5 (siRNA into DharmaFECT), mix gently by pipetting up and down, and incubate at room temperature for an additional 20 min.
  8. Add 800 μl of complete medium to the resulting mixture of step 7 (final siRNA concentration is 25 nM.).
  9. Remove culture medium from the 12-well tissue culture plate. Add the medium mixture of step 8 (total of 1 ml) to each well (see Note 4).
  10. Grow Hela cells for additional 24-48 h before mRNA analysis, or >48 h for protein analysis.
  11. Cytotoxicity should always be carefully monitored throughout the knockdown process. Experimental conditions should always be determined empirically.
  12. Each experiment should have “control groups” including Non-treated cells, Positive control siRNA (e.g., GAPD), Negative control siRNA (e.g., “Non-targeting”). Perform experiments in triplicates as a minimum.

Notes

  1. Optimal cell seeding density for each cell type should always be determined empirically. For Hela cells, the cell density should reach ~50% confluence at the beginning of the knockdown procedure.
  2. 1 μl of DharmaFECT reagent was found to yield good knockdown results.
  3. Use different DharmaFECT Transfection reagent for different cell lines. Check www.dharmacon.com for details.
  4. For steps 8-9: Alternatively, one can also replenish the original medium in the well with 800 μl of fresh complete medium, followed by evenly “dropping” the 200 μl reagent mixture of step 7 into each well.

Acknowledgments

This protocol was developed in the Department of Immunology, Scripps Research Institute, La Jolla, CA, USA and adapted from Elbashir et al. (2001), Hamilton and Baulcombe (1999) and Hannon and Rossi (2004). The work was funded by NIH grants CA079871 and CA114059, and Tobacco-Related Disease, Research Program of the University of California, 15RT-0104 to Dr. Jiing-Dwan Lee [see Chen et al. (2009)].

References

  1. Chen, Y., Lu, B., Yang, Q., Fearns, C., Yates, J. R., 3rd and Lee, J. D. (2009). Combined integrin phosphoproteomic analyses and small interfering RNA--based functional screening identify key regulators for cancer cell adhesion and migration. Cancer Res 69(8): 3713-3720.
  2. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411(6836): 494-498. 
  3. Hamilton, A. J., Baulcombe, D. C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286(5441): 950-952. 
  4. Hannon, G. J. and Rossi, J. J. (2004). Unlocking the potential of the human genome with RNA interference. Nature 431(7006): 371-378.

简介

小干扰RNA(siRNA)是一类20-25个核苷酸的双链RNA,在许多生物过程中起重要作用(Hamilton和Baulcombe,1999)。 siRNA通过"中和"靶蛋白的mRNA起作用,促进mRNA的降解,从而改变蛋白的生物效应(综述于Hannon和Rossi,2004)。 siRNA也可以改变调节RNA的细胞内水平。 在生物研究中使用siRNA操纵目的基因的表达通常被称为RNA干扰或敲除技术(Elbashir等人,2001)。 合成的siRNA是一种新兴的工具,现在广泛应用于这些研究。 不同公司采用多种算法来设计siRNA产品,其在功效,特异性和成本等方面不同。 在此使用来自Dharmacon的siGENOME SMARTpool试剂解释siRNA敲低的示例方案。

材料和试剂

  1. 人宫颈上皮癌细胞系Hela(ATCC,目录号:CCL-2 TM)
  2. Eagle's Minimum Essential Medium(ATCC,目录号:30-2003 TM)
  3. 胎牛血清(FBS)(ATCC,目录号:30-2020 TM)
  4. 5x siRNA缓冲液(GE Healthcare Dharmacon,目录号:B-002000-UB-100)
  5. DharmaFECT 1siRNA转染试剂(GE Healthcare Dharmacon,目录号:T-2001-01)
  6. siGENOME SMARTpool试剂(GE Healthcare Dharmacon)
  7. 甘油脱氢-3-磷酸脱氢酶(GAPD)(GE Healthcare Dharmacon,目录号:D-001140-01)
  8. 非靶向siRNA对照池(GE Healthcare Dharmacon,目录号:D-001206-13)

设备

  1. 12孔聚苯乙烯组织培养板(BD Biosciences,Falcon ,目录号:353043)
  2. 细胞培养孵育器:37℃和5%CO 2/h

程序

  1. 在含有10%FBS的Eagle's最低必需培养基中携带Hela细胞
  2. 胰蛋白酶消化,计数细胞,并重新饲养细胞12-16 h前击倒(见注1)。
  3. 在1x siRNA缓冲液中重悬siRNA,使终浓度达到5μM
  4. 将5μl5μMsiRNA加入到95μl无血清培养基中的低粘附管1中,轻轻点击试管或上下吹打混匀。
  5. 在另一管2中加入0.5〜5μlDharmaFECT 1试剂(见注2)至99μl无血清培养基中,轻轻敲击试管或上下吹打混匀(见注3)。
  6. 将步骤4-5中的两个管在室温下孵育5分钟
  7. 将步骤4的管1的内容物加入步骤5的管2(siRNA进入DharmaFECT),通过上下吹打轻轻混合,并在室温下再温育20分钟。
  8. 向所得的步骤7的混合物中加入800μl完全培养基(最终siRNA浓度为25nM)
  9. 从12孔组织培养板中取出培养基。向每个孔中加入步骤8的培养基混合物(总共1ml)(参见注释4)
  10. 在mRNA分析前再生长Hela细胞24-48小时,或对于蛋白质分析大于48小时
  11. 在整个敲除过程中应始终仔细监测细胞毒性。实验条件应始终由经验确定
  12. 每个实验应该具有"对照组",包括未处理的细胞,阳性对照siRNA(例如,GAPD),阴性对照siRNA(例如,"非靶向")。 至少进行三次实验。

笔记

  1. 每种细胞类型的最佳细胞接种密度应始终根据经验确定。 对于Hela细胞,在敲除程序开始时细胞密度应达到〜50%汇合
  2. 发现1μlDharmaFECT试剂产生良好的击倒结果
  3. 对不同的细胞系使用不同的DharmaFECT Transfection试剂。 有关详细信息,请访问www.dharmacon.com。
  4. 对于步骤8-9:或者,也可以用800μl新鲜的完全培养基补充孔中的原始培养基,然后将步骤7的200μl试剂混合物均匀地"滴加"到每个孔中。

致谢

该方案在美国加利福尼亚州La Jolla的Scripps研究所免疫学系中开发,并改编自Elbashir等人(2001),Hamilton和Baulcombe(1999)和Hannon和Rossi(2004) )。这项工作由NIH资助,CA079871和CA114059,加州大学的烟草相关疾病研究计划,15RT-0104给Jiing-Dwan Lee博士[见Chen等人。 )]。

参考文献

  1. Chen,Y.,Lu,B.,Yang,Q.,Fearns,C.,Yates,J.R.,3rd和Lee,J.D。(2009)。 组合整合素磷蛋白分析和基于小干扰RNA的功能筛选确定了癌细胞粘附的关键调节因子, 。 Cancer Res 69(8):3713-3720。
  2. Elbashir,S.M.,Harborth,J.,Lendeckel,W.,Yalcin,A.,Weber,K.and Tuschl,T。(2001)。 21核苷酸RNA的双链体介导培养的哺乳动物细胞中的RNA干扰。 自然 411(6836):494-498。 
  3. Hamilton,A.J.,Baulcombe,D.C。(1999)。 植物中转录后基因沉默中的一种小反义RNA。 科学 286(5441):950-952。
  4. Hannon,G.J。和Rossi,J.J。(2004)。 使用RNA干扰解锁人类基因组的潜力。 自然< em> 431(7006):371-378。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, Y. (2012). Dharmacon siRNA Transfection of HeLa Cells. Bio-protocol 2(4): e85. DOI: 10.21769/BioProtoc.85.
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