Name: a chromatin assay for human brain tissue
Uploaded: 2008-03-21T19:19:00
Description: Watch this Scientific Journal Video about A Chromatin Assay for Human Brain Tissue at JoVE.com

这项工作是从美国国立精神卫生研究所（5R01MH071476）的赠款支持。

程序： 第一日 1。均质与Douncing缓冲区50-500毫克冷冻验尸报告灰色的事情组织。 ！注意-人体组织必须谨慎处理，严格的安全条件下。它应在BSL - 2或更高的安全标准来处理。 <ol><li>以前解剖，验尸报告的大脑从-80 °，dounce Douncing缓冲区5X大脑体积，并在2.0 ml离心管地方。匹配的样品，并控制对同时处理。 </li></ol><p c...

<ol>
	<li>Huang, H. S., Matevossian, A., Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akbarian+S.+Chromatin+immunoprecipitation+in+postmortem+brain.">Akbarian S. Chromatin immunoprecipitation in postmortem brain.</a> J Neurosci Methods. 156, 284-292 .</li><li>Huang, H. S., Matevossian, A., Whittle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prefrontal+dysfunction+in+schizophrenia+involves+mixed-lineage+leukemia+1-regulated+histone+methylation+at+GABAergic+gene+promoters.">Prefrontal dysfunction in schizophrenia involves mixed-lineage leukemia 1-regulated histone methylation at GABAergic gene promoters.</a> J Neurosci. 27, 11254-11262 .</li><li>O'Neill, L. P., Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprecipitation+of+native+chromatin:+NChIP.">Immunoprecipitation of native chromatin: NChIP.</a> Methods. 31, 76-82 .</li><li>Spiteri, E., Konopka, G., Coppola, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+transcriptional+targets+of+FOXP2,+a+gene+linked+to+speech+and+language,+in+developing+human+brain.">Identification of the transcriptional targets of FOXP2, a gene linked to speech and language, in developing human brain.</a> Am J Hum Genet. 81, 1144-1157 .</li><li>Huang, H. u. a. n. g., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAD1+mRNA+expression+and+DNA+methylation+in+prefrontal+cortex+of+subjects+with+schizophrenia.">GAD1 mRNA expression and DNA methylation in prefrontal cortex of subjects with schizophrenia.</a> PLoS ONE. 2, .</li><li>Stadler, F., Kolb, G., Rubusch, L., Baker, S. P., Jones, E. G., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+methylation+at+gene+promoters+is+associated+with+developmental+regulation+and+region-specific+expression+of+ionotropic+and+metabotropic+glutamate+receptors+in+human+brain.">Histone methylation at gene promoters is associated with developmental regulation and region-specific expression of ionotropic and metabotropic glutamate receptors in human brain.</a> J Neurochem. 94, 324-336 .</li></ol>

这里列出的协议在人类大脑的组蛋白和/或DNA甲基化签名感兴趣的调查是非常有用的的，因为这些染色标记可能不容易死后文物相比，其他类型的修改，包括（组蛋白）的乙酰化和磷酸化1的， 2。死后的大脑，是适合单核小体的准备的研究，在很大程度上仍然连接到核心组蛋白的DNA，至少在与代表自溶的时间间隔（死亡和冻结/储存组织之间的时间）范围内的典型标本，几个小时至1.5天。然而，单?...

解决方案： <ul><li> nChIP Douncing缓冲液：10毫米三，MgCl 2的4毫米，1毫米氯化钙 2调整pH值至7.5 </li><li> 10X外频：50 mM的EDTA，三200毫米，500毫米氯化钠调整pH值至7.5 </li><li>低盐洗涤液：0.1％SDS，1％Triton X - 100的的，2毫米EDTA，20毫米的Tris（pH值8），150毫米氯化钠</li><li>高盐洗涤液：0.1％SDS，1％TRITON X - 100，2 mM的EDTA，20毫米的三（PH 8），500毫米氯化钠</li><li>氯化锂缓冲液：1％的IGEPAL - CA 630，1％去氧胆酸，1毫米EDTA（pH值8），10毫米三（pH值8），氯化锂0.25米</li><li> TE缓冲液（pH值8）：10毫米三，1毫米EDTA </li><li>洗脱缓冲液：0.1M碳酸氢钠，1％SDS </li><li>蛋白酶K消化缓冲液（10X）：1M的Tris，50 mM的EDTA，2％SDS，2M氯化钠调节pH至8.0 </li><li> 3 M醋酸钠：24.61 g无水醋酸钠100毫升灭菌蒸馏水（MW = 82.03）。调整pH值至5.2使用浓醋酸。 </li></ul>

a chromatin assay for human brain tissue

慢性神经精神疾病如精神分裂症，双相情感疾病和自闭症被认为是导致从遗传和环境因素可能会导致基因表达和其他分子病理学遗传学改变相结合。然而，传统上，死后大脑中的表达研究局限于基因或蛋白质的量化。在死后大脑的研究，如在自溶时间的可变性和组织integrities中遇到的限制也可能会影响到任何高阶的染色质结构研究。然而，基因组DNA的核小体的组织，包括的DNA：核心组蛋白具有约束力 - 看来主要是由不同的大脑银行提供的有代表性的样品保存。因此，它是研究在死后大脑中定义的基因位点的核心组蛋白的甲基化模式和其他共价修饰。在这里，我们提出一个简化的本地冻结（从来没有固定的）人类的大脑标本的染色质免疫沉淀（NChIP）协议。微球菌核酸酶的消化脑组织匀浆开始，NChIP由qPCR的三天之内就可以完成。这里介绍的方法应该是有用的，以澄清在正常和病变的人类大脑基因表达的表观遗传机制。

Watch this Scientific Journal Video about A Chromatin Assay for Human Brain Tissue at JoVE.com

A Chromatin Assay for Human Brain Tissue

直到最近，表达对人类大脑的研究仅限于RNA或蛋白质的定量。本文中所描述的染色质免疫沉淀技术，将有可能映射在死后大脑基因表达的组蛋白甲基化和其他后生监管。

一个人脑组织中的染色质含量

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Research

JoVE Journal

Biology

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

肌动蛋白共沉淀测定;​​ F -肌动蛋白结合蛋白分析

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

科研

生物学

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

从人死后脑组织神经元细胞核分离

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

在激活人类细胞中的基因表达的动态检测组蛋白修饰染色质免疫沉淀（ChIP）

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Chromatin Immunoprecipitation ChIP using Drosophila tissue

染色质免疫沉淀（ChIP）使用果蝇组织

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

产生和人类INO80染色质重塑复合物及其亚纯化

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

适用于比色金纳米粒子检测方法的选择结构切换适配方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...