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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.

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Dharmacon siRNA Transfection of HeLa Cells
Dharmacon siRNA 转染HeLa细胞

分子生物学 > RNA > RNA 干扰
作者: Yanling Chen
Yanling ChenAffiliation: Department of Immunology, The Scripps Research Institute, La Jolla, USA
For correspondence: ylchen@scripps.edu
Bio-protocol author page: a27
Vol 2, Iss 4, 2/20/2012, 11200 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.85

[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.

[Abstract] 小干扰RNA (siRNA)是一类20~25nt大小的双链RNA,在生物体内起着重要的作用 [1]. 人工合成的siRNAs 已经成为一个有效的工具广泛的应用于调控基因转录和翻译,它可以作用于感兴趣的靶向蛋白使其mRNA失效(“敲除”),这一过程通常被称为RNA干扰[2].RNA干扰(敲除)是一个使靶向RNA降解的复杂过程[3 & refs therein]. 对同一靶向RNA,通过不同的公司可能设计的干扰小RNA不同,这里介绍一种用Dharmacon (Thermo)的 siGENOME SMARTpool试剂介导的siRNA转染的实验方法 。

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.

耗材,试剂和仪器

 

1.       人子宫颈增殖表皮癌细胞HeLa (ATCC #CCL-2)

2.       12-孔聚苯乙烯组织培养板(BD Falcon #353043)

3.       Eagle的最低基础培养基(ATCC #30-2003)

4.       胎牛血清ATCC #30-2020)

5.       siRNA 缓冲液(5x, Dharmacon #B-002000-UB-100)

6.       DharmaFECT 1 siRNA 转染试剂 (Dharmacon #T-2001-01)

7.       siGENOME SMARTpool 试剂

8.       细胞培养箱: 37 °C  5% CO2

9.       GAPD Dharmacon #D-001140-01

10.   无靶向, Dharmacon #D-001206-13

 

步骤

 

1.        HeLa细胞在含有 Eagle最低基础培养基上培养。

2.       用胰蛋白酶作用细胞系, 对细胞计数并且在干扰前重新接种细胞1216h

注意: 每一类细胞最适合的培养浓度必须通过经验来判断。对于HeLa细胞在干扰实验的开始应该达到~50%汇合。

3.       1x siRNA缓冲液重悬siRNA 使终浓度达到5μM

4.       用一个低附着力的管子1,加 5 μl 重悬的 5 μM siRNA95 μl无血清培养基中, 轻轻的敲击或者用移液器上下吹打混匀.

5.       在管子2中加 0.5~5 μl DharmaFECT 1 试剂 (注意: 1 μl足可以达到很好的敲除效果) 99 μl 无血清培养基中, 轻轻的敲击或者用移液器上下吹打混匀.

注意:根据不同的细胞系用不同的 DharmaFECT 转染试剂. 请查询 www.dharmacon.com

6.       4&5步骤中的两个管子在室温下静置孵化5分钟。

7.       将管1中的成分加到管2 (siRNA  DharmaFECT), 轻轻的用枪吹打混匀并且在室温中静置20min

8.       800 μl 完全营养培养基到第七步骤的管子中 (最终siRNA 浓度为25 nM).

9.       12-孔聚苯乙烯组织培养板移除,加第八步骤中的混合液(共1ml)到每一个孔中。

注意: 对于步骤89,你也可以用最初的800μl新鲜的完整营养培养基补充,然后在将步骤7中的混合液加到每一个孔中。

10.   在检测mRNA前将HeLa细胞继续培养24-48小时,检测蛋白要培养多于48小时。

11.   在整个敲除实验中要小心的检测细胞毒性,应该通过经验最终确定实验条件。

12.   每一个实验都应该有对照组,包括没有处理的细胞,正对照(e.g., GAPD Dharmacon #D-001140-01),负对照siRNA (e.g., “Non-targeting,Dharmacon #D-001206-13). 将实验分为3份或者按照自己的计划分类.

 

参考文献:

 

1.        Hamilton A.J., Baulcombe D.C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286(5441): 950-2. 

2.        Elbashir S.M., Harborth J., Lendeckel W., Yalcin A., Weber K., Tuschl T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411(6836): 494-8. 

3.        Hannon G.J., Rossi J.J. (2004). Unlocking the potential of the human genome with RNA interference. Nature 431(7006): 371-8. 

 

 

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How to cite this protocol: Chen, Y. (2012). Dharmacon siRNA Transfection of HeLa Cells. Bio-protocol 2(4): e85. DOI: 10.21769/BioProtoc.85; Full Text



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