SignalSilence? EWS siRNA I

• 产品编号：CST-12175S      品牌：CST       原厂货号：12175S
• 产品分类：DNA和RNA纯化 > RNA干扰 > RNAi Oligos > siRNA
• 应用分类：

描述：

来自Cell Signaling Technology （CST）的SignalSilence® EWS siRNA I可以通过RNA干扰特异性抑制EWS的表达。这种方法可以通过将双链RNA分子传递到细胞内从而使基因表达有选择的沉默。来自CST的所有的SignalSilence^® siRNA产品都是经过内部严格检测的，并且通过Western blot 分析证明确实能够减少目的蛋白的表达。通过三苯甲基分析每个碱基以监测寡核苷酸的合成，确保合适的配对效率。随后寡核苷酸通过亲和固相萃取法纯化。退火的RNA双链通过质谱分析来证实其精确的组成。每一批产品都通过质谱分析与前面的产品进行比较，来保证不同批次之间的最大一致性。CST建议使用100 nM SignalSilence® EWS siRNA I进行转染，48到72小时后对细胞进行裂解。转染步骤按照转染试剂说明书提供的步骤进行。遇到任何使用方面的问题，请随时联系CST。每小瓶可供100次转染，每次转染量相当于在转染24孔板时，每孔总体积为300μl培养基中siRNA的终浓度为100nM。尤文氏肉瘤（EWS）蛋白是多功能FET（FUS、 EWS和TAF15）蛋白家族的成员（1,2）。这些蛋白是RNA和DNA结合蛋白，被认为对转录调控和RNA加工都有重要作用。在尤文氏肉瘤肿瘤家族中发现，EWS可以与多种E-twenty six (ETS)家族转录因子，最常见的是Fli-1形成融合蛋白（1-4）。EWS的氨基末端含有一个转录激活结构域，与ETS转录因子的DNA结合结构域融合在一起，从而引起目的基因的异常表达（1-5）。EWS可以通过TFIID以及RNA聚合酶II 亚单位与转录起始复合物相互作用，与Brn3A 和 CBP/p300等转录因子也是一样的，这都暗示着EWS在转录调控中的作用（1,6-9）。此外EWS还能与剪接机器中的多种成分产生相互作用，参与RNA 加工（1,10-12）。EWS能够从转录激活和mRNA加工水平上调控Cyclin D1的表达，而Cyclin D1可以控制细胞周期中G1-S期转换。EWS-Fli-1融合蛋白已经被证明能够促进尤文氏肉瘤细胞中cyclin D1b 剪接异构体的表达（13）。另外，EWS可调控基因中DNA损伤诱导的选择性剪接作用从而参与DNA修复以及应激反应，并且是DNA损伤后细胞生存所需要的。（14）。与这些结果一致的是，EWS敲除小鼠会呈现出对电离辐射过敏和过早细胞衰老，这些都暗示着，EWS在同源重组和基因组稳定性维持中的作用（15）。

1. Law, W.J. et al. (2006) Brief Funct Genomic Proteomic 5, 8-14.
2. Kovar, H. (2011) Sarcoma 2011, 837474.
3. Delattre, O. et al. (1992) Nature 359, 162-5.
4. May, W.A. et al. (1993) Mol Cell Biol 13, 7393-8.
5. Sorensen, P.H. et al. (1994) Nat Genet 6, 146-51.
6. Bertolotti, A. et al. (1996) EMBO J 15, 5022-31.
7. Bertolotti, A. et al. (1998) Mol Cell Biol 18, 1489-97.
8. Araya, N. et al. (2003) J Biol Chem 278, 5427-32.
9. Thomas, G.R. and Latchman, D.S. Cancer Biol Ther 1, 428-32.
10. Chansky, H.A. et al. (2001) Cancer Res 61, 3586-90.
11. Yang, L. et al. (2000) J Biol Chem 275, 37612-8.
12. Knoop, L.L. and Baker, S.J. (2001) J Biol Chem 276, 22317-22.
13. Sanchez, G. et al. (2008) Proc Natl Acad Sci U S A 105, 6004-9.
14. Paronetto, M.P. et al. (2011) Mol Cell 43, 353-68.
15. Li, H. et al. (2007) J Clin Invest 117, 1314-23.

原厂资料：

Homology

Species predicted to react based on 100% sequence homology: Mouse, Rat, Monkey

Specificity / Sensitivity

SignalSilence® EWS siRNA I inhibits human, mouse, rat, and monkey EWS expression.

Description

SignalSilence® EWS siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit EWS expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.

Quality Control

Oligonucleotide synthesis is monitored base by base through trityl analysis to ensure appropriate coupling efficiency. The oligo is subsequently purified by affinity-solid phase extraction. The annealed RNA duplex is further analyzed by mass spectrometry to verify the exact composition of the duplex. Each lot is compared to the previous lot by mass spectrometry to ensure maximum lot-to-lot consistency.

Directions for Use

CST recommends transfection with 100 nM SignalSilence® EWS siRNA I 48 to 72 hours prior to cell lysis. For transfection procedure, follow protocol provided by the transfection reagent manufacturer. Please feel free to contact CST with any questions on use. 
 Each vial contains the equivalent of 100 transfections, which corresponds to a final siRNA concentration of 100 nM per transfection in a 24-well plate with a total volume of 300 μl per well.

Background

The Ewing sarcoma (EWS) protein is a member of the multifunctional FET (FUS, EWS, and TAF15) family of proteins (1,2). These proteins are RNA and DNA binding proteins that are thought to be important for both transcriptional regulation and RNA processing. EWS can be found as part of a fusion protein with various E-twenty six (ETS) family transcription factors, most commonly Fli-1, in the Ewing sarcoma family of tumors (1-4). The amino terminus of the EWS protein, containing the transcriptional activation domain, is fused to the DNA binding domain of the ETS transcription factor, causing aberrant expression of target genes (1-5). EWS interacts with the transcription initiation complex via TFIID and RNA polymerase II subunits, as well as transcriptional regulators, such as Brn3A and CBP/p300, which suggests a role for EWS in transcriptional regulation (1,6-9). EWS also interacts with multiple components of the splicing machinery, implicating a role for EWS in RNA processing (1,10-12). EWS regulates the expression of cyclin D1, which controls G1-S phase transition during the cell cycle, at the level of transcriptional activation and mRNA splicing. The EWS-Fli-1 fusion protein has been shown to promote the expression of the cyclin D1b splice variant in Ewing sarcoma cells (13). In addition, EWS regulates the DNA damage-induced alternative splicing of genes involved in DNA repair and stress response and is required for cell viability upon DNA damage (14). Consistent with these results, EWS knockout mice display hypersensitivity to ionizing radiation and premature cellular senescence, suggesting a role for EWS in homologous recombination and maintenance of genomic stability (15).

1. Law, W.J. et al. (2006) Brief Funct Genomic Proteomic 5, 8-14.
2. Kovar, H. (2011) Sarcoma 2011, 837474.
3. Delattre, O. et al. (1992) Nature 359, 162-5.
4. May, W.A. et al. (1993) Mol Cell Biol 13, 7393-8.
5. Sorensen, P.H. et al. (1994) Nat Genet 6, 146-51.
6. Bertolotti, A. et al. (1996) EMBO J 15, 5022-31.
7. Bertolotti, A. et al. (1998) Mol Cell Biol 18, 1489-97.
8. Araya, N. et al. (2003) J Biol Chem 278, 5427-32.
9. Thomas, G.R. and Latchman, D.S. Cancer Biol Ther 1, 428-32.
10. Chansky, H.A. et al. (2001) Cancer Res 61, 3586-90.
11. Yang, L. et al. (2000) J Biol Chem 275, 37612-8.
12. Knoop, L.L. and Baker, S.J. (2001) J Biol Chem 276, 22317-22.
13. Sanchez, G. et al. (2008) Proc Natl Acad Sci U S A 105, 6004-9.
14. Paronetto, M.P. et al. (2011) Mol Cell 43, 353-68.
15. Li, H. et al. (2007) J Clin Invest 117, 1314-23.

注意事项：

Storage: EWS siRNA I is supplied in RNAse-free water. Aliquot and store at -20ºC.