SimpleChIP? Human NR1D1 Promoter Primers

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• 产品分类：PCR/RT-PCR/qRT-PCR > 其它 > 引物 > 其他引物与寡核苷酸
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描述：

Human NR1D1 Promoter Primers包含一对正向和反向PCR引物，该引物特异性针对人NR1D1启动子，能用来扩增用染色质免疫沉淀法（ChIP）分离的DNA。该引物已经用SYBR® Green quantitative real-time PCR进行优化，并且用SimpleChIP® Enzymatic Chromatin IP Kits #9002 and #9003 和CST公司的ChIP验证抗体进行测试。The NR1D1 gene编码转录抑制因子Rev-erbα，它是一种孤儿核受体，在许多组织，器官中调控细胞增殖，分化，与生理周期。标签适当数量的PCR管或与实时定量PCR仪兼容的PCR板。PCR反应应重复执行,应该包括一个管没有DNA作为对照,和一系列不同浓度2%的总染色质DNA(1:5,1:25,1:125),用于创建一个标准曲线并明确扩增效率。2。在每一个管或PCR板孔中添加2μl合适的ChIP DNA样本。3。准备一个PCR反应体系如下所述。考虑到体积的损失可额外添加足够两个反应的试剂。在每个PCR反应管或PCR板孔中添加18μl PCR反应混合体。一个 PCR反应(20μl)的试剂体积< Nuclease-free水6μl 5μM SimpleChIP^® Primers 2 2μl 2 x SYBR^® Green Reaction Mix 10μl. 开始PCR反应程序如下:初始变性:95°C 3分钟。变性:95°C,持续15秒。退火和延伸:引物特异性温度，60秒。重复步骤b和c总共40周期。使用实时PCR仪提供的软件分析定量PCR结果。染色质免疫沉淀（chromatin immunoprecipitation ，ChIP）分析是一个有力且通用的技术，能用来探测细胞内天然染色质环境中的蛋白质-DNA相互作用(1,2)。这种分析能用于鉴定多种与基因组某个特定区域相关的蛋白，或者鉴定基因组中许多与某种特殊蛋白结合的区域(3-6)。ChIP能确定募集到基因启动子的多种蛋白的特异的顺序，或者“度量”整个基因位点特定的组蛋白修饰的相对数量 (3,4)。除了组蛋白外，ChIP还能用于分析转录因子与辅助因子、DNA复制因子和DNA修复蛋白的结合。当进行ChIP 分析时，细胞首先用甲醛固定，甲醛是一种可逆的蛋白质-DNA交联剂，它可以“保存”在细胞内进行的蛋白质-DNA相互作用 (1,2)。然后将细胞裂解，收集染色质并用超声波或酶消化成片段，片段的染色质用特异性针对特定蛋白或组蛋白修饰的抗体进行免疫沉淀。任何与蛋白或有关组蛋白修饰相关的DNA序列都会作为交联染色质复合物的一部分与其共沉淀，并且该DNA序列的相对数量会在免疫选择过程中增加。在免疫沉淀之后，蛋白质-DNA交联将逆转，DNA就被纯化了。标准PCR或实时定量PCR经常用于测量进行蛋白质特异免疫沉淀后特定DNA序列的富集量(1,2)。另外，ChIP分析能选择性地与基因组微阵列技术 (ChIP on chip)、高通量测序技术(ChIP-Seq)或克隆策略相结合，它们都能进行蛋白质-DNA相互作用和组蛋白修饰的全基因组分析(5-8)。经过实时定量PCR优化，SimpleChIP® primers是ChIP分离的DNA扩增的最佳引物，并且提供用于确认一个成功的ChIP实验的阳性对照和阴性对照。

1. Orlando, V. (2000) Trends Biochem Sci 25, 99-104.
2. Kuo, M.H. and Allis, C.D. (1999) Methods 19, 425-33.
3. Agalioti, T. et al. (2000) Cell 103, 667-78.
4. Soutoglou, E. and Talianidis, I. (2002) Science 295, 1901-4.
5. Mikkelsen, T.S. et al. (2007) Nature 448, 553-60.
6. Lee, T.I. et al. (2006) Cell 125, 301-13.
7. Weinmann, A.S. and Farnham, P.J. (2002) Methods 26, 37-47.
8. Wells, J. and Farnham, P.J. (2002) Methods 26, 48-56.

原厂资料：

Description

SimpleChIP^® Human NR1D1 Promoter Primers contain a mix of forward and reverse PCR primers that are specific to a region of the human nuclear receptor subfamily 1, group D, member 1 (NR1D1) promoter. These primers can be used to amplify DNA that has been isolated using chromatin immunoprecipitation (ChIP). Primers have been optimized for use in SYBR^® Green quantitative real-time PCR and have been tested in conjunction with SimpleChIP^® Enzymatic Chromatin IP Kits #9002 and #9003 and ChIP-validated antibodies from Cell Signaling Technology^®. The NR1D1 gene encodes for the transcriptional repressor Rev-erbα, an orphan nuclear receptor that regulates cell proliferation, differentiation, and circadian rhythms in multiple tissues and organ systems.

Directions for Use

1. Label the appropriate number of PCR tubes or PCR plates compatible with the model of real-time PCR machine to be used. PCR reactions should be performed in duplicate and should include a tube with no DNA to control for contamination, and a serial dilution of a 2% total input chromatin DNA (undiluted, 1:5, 1:25, 1:125), which is used to create a standard curve and determine amplification efficiency.2. Add 2 μl of the appropriate ChIP DNA sample to each tube or well of the PCR plate.

3. Prepare a master PCR reaction mix as described below. Add enough reagents for two extra reactions to account for loss of volume. Add 18 μl of the master PCR reaction mix to each PCR reaction tube or well of the PCR plate.

Reagent Volume for 1 PCR Reaction (20 μl)

Nuclease-free H2O 6 μl

5 μM SimpleChIP^® Primers 2 μl

2X SYBR^® Green Reaction Mix 10 μl

4. Start the following PCR reaction program:

a. Initial Denaturation: 95°C for 3 min.

b. Denaturation: 95°C for 15 sec.

c. Anneal and Extension: Primer-specific temp. for 60 sec.

d. Repeat steps b and c for a total of 40 cycles.

5. Analyze quantitative PCR results using software provided with the real-time PCR machine.

Background

The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This assay can be used to either identify multiple proteins associated with a specific region of the genome or to identify the many regions of the genome bound by a particular protein (3-6). ChIP can be used to determine the specific order of recruitment of various proteins to a gene promoter or to "measure" the relative amount of a particular histone modification across an entire gene locus (3,4). In addition to histone proteins, the ChIP assay can be used to analyze binding of transcription factors and co-factors, DNA replication factors, and DNA repair proteins. When performing the ChIP assay, cells are first fixed with formaldehyde, a reversible protein-DNA cross-linking agent that "preserves" the protein-DNA interactions occurring in the cell (1,2). Cells are lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion. Fragmented chromatin is then immunoprecipitated with antibodies specific to a particular protein or histone modification. Any DNA sequences that are associated with the protein or histone modification of interest will co-precipitate as part of the cross-linked chromatin complex and the relative amount of that DNA sequence will be enriched by the immunoselection process. After immunoprecipitation, the protein-DNA cross-links are reversed and the DNA is purified. Standard PCR or quantitative real-time PCR are often used to measure the amount of enrichment of a particular DNA sequence by a protein-specific immunoprecipitation (1,2). Alternatively, the ChIP assay can be combined with genomic tiling micro-array (ChIP on chip) techniques, high throughput sequencing (ChIP-Seq), or cloning strategies, all of which allow for genome-wide analysis of protein-DNA interactions and histone modifications (5-8). SimpleChIP^® primers have been optimized for amplification of ChIP-isolated DNA using real-time quantitative PCR and provide important positive and negative controls that can be used to confirm a successful ChIP experiment.

1. Orlando, V. (2000) Trends Biochem Sci 25, 99-104.
2. Kuo, M.H. and Allis, C.D. (1999) Methods 19, 425-33.
3. Agalioti, T. et al. (2000) Cell 103, 667-78.
4. Soutoglou, E. and Talianidis, I. (2002) Science 295, 1901-4.
5. Mikkelsen, T.S. et al. (2007) Nature 448, 553-60.
6. Lee, T.I. et al. (2006) Cell 125, 301-13.
7. Weinmann, A.S. and Farnham, P.J. (2002) Methods 26, 37-47.
8. Wells, J. and Farnham, P.J. (2002) Methods 26, 48-56.

注意事项：

Storage: Supplied in nuclease-free water at a concentration of
5 μM (each primer is at a final concentration of 5 μM). Store at
-20°C.