SignalSilence? Vitamin D3 Receptor siRNA I

• 产品编号：CST-12719S      品牌：CST       原厂货号：12719S
• 产品分类：DNA和RNA纯化 > RNA干扰 > RNAi Oligos > siRNA
• 应用分类：

描述：

制维生素D3受体的表达，使用这种方法可以将双链RNA分子转至细胞内，选择性沉默基因的表达。所有CST SignalSilence® siRNA产品都经过严格的内部测试，并用western方法检测证明可以降低靶蛋白的表达。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，该寡核苷酸通过亲和固相萃取纯化。退火的RNA双链进一步通过质谱法进行分析，验证双链的确切成分。通过质谱，将每批结果与之前的批次比较，以确保批次间最大的一致性。CST建议在细胞裂解前48-72h，用100 NM SignalSilence® Vitamin D3 Receptor siRNA I进行转染。转染程序参照转染试剂制造商所提供的操作指南。在使用上有任何问题，请随时联系CST。每管含有相当于100个转染的量，这相当于每个24孔板，每次转染用100 nM的终浓度siRNA，每孔总体积为300μl。虽然最初根据维生素D3受体在钙和骨稳态中的作用对其进行认定，但现在在人体几乎每个组织中，维生素D3受体（VDR/NR1I1）及其配体1-α，25 - 二羟胆钙化醇[1α，25（OH）2D3]发挥的生物效应被识别。维生素D的信号靶标包括中枢神经系统、皮肤、免疫系统、内分泌腺体、肾和结肠。在细胞水平上，维生素D信号影响正常和转化细胞的增殖、分化和凋亡。类固醇受体基因家族中，VDR属于NR1I亚科，该亚科还包括NR1I2/PXR和NR1I3/CAR。人维生素D受体基因由编码全长VDR蛋白6个域（AF）的11个外显子组成，其中包括一个N-末端的双锌指结构DNA结合域，一个C-末端配体结合活性域和广泛链接这两个功能域的非结构化区域（1）。1α，25（OH）2D3结合激素配体结合域，由PKC磷酸化DNA结合结构域的丝氨酸（51位）（2）和酪蛋白激酶II磷酸化铰链区的丝氨酸（208位）（3），使VDR稳定。VDR与维甲酸受体（RXR）通过二聚化联合。 1α，25（OH）2D3 VDR-RXR复合物结合维生素D3响应元件(VDREs)通过DNA结合结构域启动靶基因。VDR中配体诱导的构象变化，导致辅阻遏物、维甲酸和甲状腺激素受体沉默介质(SMRT)的降解，并允许VDR活化功能转录激活域（AF2）与转录共激活因子的相互作用（1）。研究表明，可变VDR的表达与癌症不同类型或阶段有关，这可能是由1α, 25(OH)2D3信号中组织类型的变异造成的。研究表明，在结肠癌的情况下，VDR在增生性结肠息肉和早期肿瘤中表达相对较高，但在以后的阶段和低分化肿瘤中表达减少。多项研究表明，1α，25（OH）2D3可能是作为一种治疗型抗癌药物开发有吸引力的靶点（4,5）。

1. Haussler, M.R. et al. (1998) J Bone Miner Res 13, 325-49.
2. Hsieh, J.C. et al. (1991) Proc Natl Acad Sci U S A 88, 9315-9.
3. Jurutka, P.W. et al. (1993) J Biol Chem 268, 6791-9.
4. Matusiak, D. et al. (2005) Cancer Epidemiol Biomarkers Prev 14, 2370-6.
5. Deeb, K.K. et al. (2007) Nat Rev Cancer 7, 684-700.

原厂资料：

Description

SignalSilence® Vitamin D3 Receptor siRNA I from Cell Signaling Technology (CST) allows the researcher to specifically inhibit vitamin D3 receptor 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® Vitamin D3 Receptor 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

Although originally identified based on their roles in calcium and bone homeostasis, the vitamin D3 receptor (VDR/NR1I1) and its ligand 1-α, 25-dihydroxycholecalciferol [1α, 25(OH)2D3] are now recognized to exert biological effects in almost every tissue of the human body. Targets for vitamin D signaling include the central nervous system, skin, immune system, endocrine glands, kidney, and colon. At the cellular level, vitamin D signaling affects proliferation, differentiation, and apoptosis of both normal and transformed cells. Within the steroid receptor gene family, VDR belongs to the NR1I subfamily that also includes NR1I2/PXR and NR1I3/CAR. The human VDR gene is composed of 11 exons that encode six domains (A-F) of the full length VDR protein, which includes an N-terminal dual zinc finger DNA binding domain, a C-terminal ligand-binding activity domain, and an extensive unstructured region that links the two functional domains together (1). Upon 1α, 25(OH)2D3 binding to the hormone ligand-binding domain, VDR is stabilized by the phosphorylation of Ser51 in the DNA-binding domain by PKC (2), and Ser208 in the hinge region by casein kinase II (3). VDR associates with the retinoic acid receptor (RXR) through dimerization domains. The 1α, 25(OH)2D3-VDR-RXR complex binds to the vitamin D response elements (VDREs) in the promoters of target genes through the DNA-binding domain. Ligand-induced conformation changes in VDR results in the dissociation of the co-repressor, silencing-mediator for retinoid and thyroid hormone receptors (SMRT), and allows interaction of the VDR activation function (AF2) transactivation domain with transcriptional coactivators (1). 
 Studies have shown that variable VDR expression is associated with different forms or stages of cancer and likely results from tissue-type variation in 1α, 25(OH)2D3 signaling. In the case of colon cancer, research indicates that VDR expression is relatively higher in hyperplastic colon polyps and during early tumorigenesis but diminishes in later stage, poorly differentiated tumors. Multiple studies suggest that 1α, 25(OH)2D3 may be an attractive target for development as a therapeutic anticancer agent (4,5) .

1. Haussler, M.R. et al. (1998) J Bone Miner Res 13, 325-49.
2. Hsieh, J.C. et al. (1991) Proc Natl Acad Sci U S A 88, 9315-9.
3. Jurutka, P.W. et al. (1993) J Biol Chem 268, 6791-9.
4. Matusiak, D. et al. (2005) Cancer Epidemiol Biomarkers Prev 14, 2370-6.
5. Deeb, K.K. et al. (2007) Nat Rev Cancer 7, 684-700.

注意事项：

Storage: Vitamin D3 Receptor siRNA I is supplied in RNAse-free water. Aliquot and store at -20ºC.