RNA纯化的染色质隔离(ChIRP)探针
ChIRP Probes in Plates
ChIRP:一种快速发现与RNA结合的DNA和蛋白质相互作用的技术。
技术原理:通过设计生物素或链霉亲和素标记的探针,把目标RNA拉下来以后,与其共同作用的DNA染色体片段就会附着在磁珠上,更后通过qRT-PCR、测序、免疫印迹或质谱分析等确定目的RNA、DNA和蛋白。
在LncRNA研究中的应用:该技术能够以高灵敏度和低背景噪音找出染色质上与LncRNA结合的基因组结合位点,绘制染色质基因协同表达图谱,检测与RNA协同表达的基因,从而了解LncRNA在染色质调控中的作用。
ChIRP is a rapid technique to discover RNA-associated DNA sequences and map genomic binding sites of chromatin associated long noncoding RNAs (lncRNAs) with high sensitivity and low background. This method allows you to map which chromatin regions come together for coordinated expression, determine which genes are expressed together, and learn the role of lncRNA in chromatin regulation. Target lncRNAs are affinity captured using antisense-oligos, designed by our ChIRP probe designer, and the lncRNA-associated DNA chromatin is sequenced to create a sequencing library. With the DNA sequence data, it is possible to generate a genomic binding site map at a resolution of several hundred bases. ChIRP oligo sets can be designed at LGC Biosearch's dedicated web-based designer and ordered directly. The oligos are delivered in plates so that they may be combined into suitable sets. We recommend buffers and other reagents for the complete ChIRP method provided by EMD/Millipore. LGC Biosearch Technologies and EMD/Millipore are exclusively licensed to provide ChIRP probe sets.
试验流程:
①探针设计:设计反义寡核苷酸探针,20nt 左右;
②细胞培养:ChiRP实验需要的细胞数,即2×107;采用的是标准培养基;
③细胞交联及收集细胞质粒;
④交联后DNA的超声破碎;
⑤生物素标记的DNA探针与RNA杂交;
⑥ChIRP;
⑦RNA、DNA及核酸结合蛋白提取,分析实验数据。
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