通过赠款，这项工作是由威康信托基金资助的APM [076566/Z/05/Z]，JCK [074318]和威康信托中心人类遗传学的核心奖[075491/Z/04]。

1）细胞培养<ol><li>选择感兴趣的基因表达的细胞类型。 </li><li>选择DNA风险的变种和转录编码内感兴趣的基因多态性（图1）杂合子的细胞株。 </li><li>文化所选单元格的行根据供应商的规格，并准备分别为2 × 10厘米的培养皿中，DNA和RNA的提取。 </li><li>当细胞达到80％汇合时启动程序，收获细胞，DNA和RNA的隔离遵循下述步骤或使用市售的试剂盒。 </li></ol> 2）DNA的提取<ol><li>从盘取出介质，用PBS清洗，并添加到菜500μL裂解液（0.6％SDS，氯化钠100毫米，50毫米的Tris，20 mM的EDTA和50μg/ mL的RNA酶A）。 </li><li>岩石板，轻轻地在室温下20分钟。 </li><li>在37 ° C过夜，刮的离心管中，并培育成的细胞裂解液。 </li><li>加入蛋白酶K至终浓度为100微克/毫升和37℃过夜孵育。 </li><li>与等体积酚 - 氯仿的pH值8，重复提取DNA，然后与等体积的氯仿/异戊醇提取。 </li><li> 1 / 10体积3 M氯化钠和2.5卷100％的乙醇沉淀的DNA。 </li><li>留在冰浴5分钟，然后在30分钟13000转，旋转4 ° C。 </li><li>用70％乙醇清洗DNA，使空气干燥，在200μL的TE悬浮。 </li><li>在65℃孵育10分钟，然后在室温下2天离开。 </li><li>保存在4 ° C或长期在-20 ° C </li></ol> 3）RNA的提取<ol><li>从盘取出介质，用PBS清洗，添加1毫升的Trizol，并培育在室温下10分钟。 </li><li>刮下的细胞，吸离心管几次，转让和孵育5分钟。 </li><li>以10,000 rpm 10分钟旋转4 ° C。 </li><li>转移在一个新的管的上清，加入200μl氯仿。 </li><li>猛烈摇动并以10,000 rpm 10分钟旋转4 ° C。 </li><li>在一个新的管水阶段转移和70％的乙醇添加量相等。 </li><li>负载的解决方案，以1或2（上卷而定）的RNeasy列和旋转的最大速度为30秒。 </li><li>从这里遵循的RNeasy试剂​​盒（Qiagen）的指令。 </li></ol> 4）核酸样品制备<ol><li>量化的DNA和RNA的浓度，使用NanoDrop分光光度计（Thermo Scientific的）。 </li><li>准备从500-1000吴RNA根据制造商的指示使用随机decamers和标三反转录酶（Invitrogen公司）的cDNA。 </li></ol> 5）PCR引物延伸实验<ol><li>设计两对PCR引物，每个DNA分子标记，基因组DNA扩增的一对，另一对cDNA扩增（图2）。广场正向和反向在不同的外显子cDNA扩增引物，以避免基因污染。设计引物，在100-500 bp的长度范围内获得的PCR产物。 </li><li>准备10μL包括0.5 U Immolase TAQ（Bioline）0.8毫米dNTPs，MgCl 2的 2毫米，0.2米的引物和20纳克cDNA或DNA的PCR反应。每个样品的扩增4个独立的反应。 </li><li>每个引物对保持线性相位的扩增循环数的优化条件下运行的PCR反应。 </li><li>取出孵化与核酸外切酶的PCR产物，PCR引物和dNTPs过剩我和虾碱性磷酸酶（SAP）的，在37 ° 20分钟。 </li><li>现货在384孔板每两次PCR产物，因此，8测量每个样品。 </li><li>设计与实验设计（Sequenom公司）软件的延伸引物，使相同的引物可用于基因组和cDNA PCR产物。引物长度在14-18 bp和延伸组合，包括3ddNTPs组合（图2）的范围内指定。 </li><li>开展引物延伸反应和MALDI-TOF/MS分析，根据制造商的指示使用MassARRAY系统（Sequenom公司）。 </li><li>一个典型的引物延伸反应，包括2分钟94步° C，5秒94 ° C，40个周期为5秒，5秒，52℃72℃ </li><li>引物延伸产品SpectroCLEAN树脂清洗，并转移到芯片（芯片）SpectroPOINT纳升级饮水机。 </li><li>扩展的寡核苷酸使用SpectroREADER质谱仪的MALDI - TOF量化。 </li></ol> 6）统计分析<ol><li>检查MALDI-TOF/MS与实验的总质量MassARRAY导电型测量仪软件结果。 </li><li>提取Allelotyping的报告，其中包括峰面积数据。 </li><li>对于每个单独的频谱计算等位基因1和等位基因2（峰面积之比）的峰面积之间的比率。 </li><li>对于每个cDNA样本推导出一个峰面积比值的均值和标准差，使用相应的功能，在Excel中，横跨8测量。 </li><li>推导出整个基因组DNA的8 measurments相同的平均峰面积之比。 </li><li>由基因组的平均比率评估偏离预期的1:1的比例分成的cDNA的平均比例。 </li></ol> 7）实验控制<ol><li>为了排除实验文物，至少有三次重复实验。 </li><li>只要有可能，额外的DNA分子标记的设计分析。 </li><li>设计多重PCR引物对和延伸引物退火正向和反向链。 </li><li>每当可能的测试，作为阴性对照细胞株，为DNA分子标记杂合，但不进行风险与表型相关的DNA变异（图1和图3。） </li></ol> 8）代表性的成果等位基因特异性表达分析的一个例子是在质谱痕迹的例子图3所示。该图显示了从4等位基因特异性引物延伸产品4种不同的模板进行分析的结果。顶部的图形表示，从两个不同的细胞系，这都是一个转录的编码基因多态性杂合子基因组DNA的分析得到的结果。然而，只有细胞系，一个是风险的DNA变异（图1）杂合子。较低的图形代表从相同的细胞系产生的cDNA进行分析得出的结果。作为一个实验神器的第一个高峰期总是高于的第二个高峰期，即使在基因组分析。因此，从基因组分析的结果用于正常化的cDNA数据。正常化后，等位基因的比例从1细胞株显着不同（其中的第二个高峰期出现低比其他光谱），虽然这是非常密切的细胞株为1 B.数据表明，细胞系一个带有DNA的变种，测量的第二个高峰期，从而降低了在分析基因表达的等位基因的阶段。细胞B线提供了十分便利的阴性对照。 <img src="/files/ftp_upload/2279/2279fig1.jpg" alt="图1" /> 图1。 A和B细胞株细胞系的选择策略 。编码一个转录标记（三角形）的杂合子，但只有一个是对风险的DNA变异（圆圈）杂合子的细胞系。蓝色的风险变异等位基因关联（直接的效果，或在与实际的功能变异密切相关，因为）的转录水平较低。 <img src="/files/ftp_upload/2279/2279fig2.jpg" alt="图2" /> 图2。等位基因特异性引物延伸实验。这个数字表明双方的cDNA和基因组DNA（左）（右）模板进行的实验过程的工作流程。 PCR引物（灰色矩形）放大一个杂合的编码基因多态性（三角形）。为了避免基因污染PCR引物退火序列放置在不同的外显子（淡蓝色酒吧）cDNA为模板进行扩增时。 PCR产物为模板，然后用一个引物延伸反应进行扩展底漆，退火一个基地旁边的多态性，三个双脱氧适当搭配（ddNTPs）终结符（正方形）和一个脱氧核苷酸（圈），以延长一个或两个的基础上底漆。所产生的引物延伸产品，允许不同的分离和MALDI - TOF质谱定量群众。相应的蓝色基因的DNA标记的产品的数量是相对较低的红色等位基因。 <img src="/files/ftp_upload/2279/2279fig3.jpg" alt="图3" /> 图3。一个等位基因特异性表达检测结果。数字显示质谱分析进行基因组DNA和细胞株的基因A和细胞B线（图1） 。

<ol>
	<li>Cookson, W., Liang, L., Abecasis, G., Moffatt, M., Lathrop, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+complex+disease+traits+with+global+gene+expression.">Mapping complex disease traits with global gene expression.</a> Nat Rev Genet. 10, 184-194 (2009).</li><li>Singer-Sam, J., LeBon, J. M., Dai, A., Riggs, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive,+quantitative+assay+for+measurement+of+allele-specific+transcripts+differing+by+a+single+nucleotide.">A sensitive, quantitative assay for measurement of allele-specific transcripts differing by a single nucleotide.</a> PCR Methods Appl. 1, 160-163 (1992).</li><li>Yan, H., Yuan, W., Velculescu, V. E., Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+variation+in+human+gene+expression.">Allelic variation in human gene expression.</a> Science. 297, 1143-11 (2002).</li><li>Ding, C., Cantor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+gene+expression+analysis+technique+using+competitive+PCR+and+matrix-assisted+laser+desorption+ionization+time-of-flight+MS.">A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS.</a> Proc Natl Acad Sci U S A. 100, 3059-3064 (2003).</li><li>Gabriel, S., Ziaugra, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNP+genotyping+using+Sequenom+MassARRAY+7K+platform.">SNP genotyping using Sequenom MassARRAY 7K platform.</a> Curr Protoc Hum Genet Chapter. Chapter 2 (Unit 2), 12-12 (2004).</li><li>Lo, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allelic+variation+in+gene+expression+is+common+in+the+human+genome.">Allelic variation in gene expression is common in the human genome.</a> Genome Res. 13 (8), 1855-1862 (2003).</li><li>Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression-based+discovery+of+variation+in+the+human+glutathione+S-transferase+M3+promoter+and+functional+analysis+in+a+glioma+cell+line+using+allele-specific+chromatin+immunoprecipitation.">Expression-based discovery of variation in the human glutathione S-transferase M3 promoter and functional analysis in a glioma cell line using allele-specific chromatin immunoprecipitation.</a> Cancer Res. 65, 99-104 (2005).</li><li>Knight, J. C., Keating, B. J., Rockett, K. A., Kwiatkowski, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+characterization+of+regulatory+polymorphisms+by+allele-specific+quantification+of+RNA+polymerase+loading.">In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading.</a> Nat Genet. 33 (4), 469-475 (2003).</li><li>Pastinen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+genetic+and+epigenetic+variation+affecting+human+gene+expression.">A survey of genetic and epigenetic variation affecting human gene expression.</a> Physiol Genomics. 16 (2), 184-193 (2004).</li><li>Francks, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+77-kilobase+region+of+chromosome+6p22.2+is+associated+with+dyslexia+in+families+from+the+United+Kingdom+and+from+the+United+States.">A 77-kilobase region of chromosome 6p22.2 is associated with dyslexia in families from the United Kingdom and from the United States.</a> Am J Hum Genet. 75 (6), 1046-1058 (2004).</li><li>Paracchini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromosome+6p22+haplotype+associated+with+dyslexia+reduces+the+expression+of+KIAA0319,+a+novel+gene+involved+in+neuronal+migration.">The chromosome 6p22 haplotype associated with dyslexia reduces the expression of KIAA0319, a novel gene involved in neuronal migration.</a> Hum Mol Genet. 15 (10), 1659-1666 (2006).</li></ol>

这种方法使体内等位基因特异性转录水平的差异，评估评价疾病相关基因表达的DNA变异的影响。在这个协议中的相对转录丰度的MALDI - TOF，但其他技术允许，如TaqMan探针6,7，等位基因特异的定量测量，都可以使用。这种方法的主要限制是转录编码标记的可用性。这里描述的方法原则的基础上，但利用不同类别的多态性，已描述。 haploChip检测措施，使用1 KB的转录的开始或结束一个基因，这将是在与特定RNA 聚合酶 II 8的抗体染色质免疫沉淀隔离材料的网站内的标记的等位基因特异性表达。此外，内含子单核苷酸多态性模板时，可以使用异RNA 的 9 。 这里描述具体的协议，与haploChip检测相结合，已成功地用于确定为读写障碍的候选基因（或阅读障碍）。最初遗传关联研究，发现与诵读困难和这是跨越3个基因的10相关的DNA序列。等位基因特异的基因表达检测结果显示，在存在的诵读困难相关的DNA变异的表达变化，发现只有三个基因中的一个，但不是其他两个11。

<table><tbody><tr><td>PBS</td><td>Sigma-Aldrich</td><td>P-4417-100TAB</td><td></td></tr><tr><td>SDS</td><td>Bio-Rad</td><td>161-0416</td><td></td></tr><tr><td>NaCl</td><td>VWR international</td><td>27788.366</td><td></td></tr><tr><td>Tris</td><td>Sigma-Aldrich</td><td>T1503-1KG</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E5134-500G</td><td></td></tr><tr><td>RNase A</td><td>Qiagen</td><td>19101</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma-Aldrich</td><td>P2308-500MG</td><td></td></tr><tr><td>Phenol: chloroform</td><td>Sigma-Aldrich</td><td>77617</td><td></td></tr><tr><td>Chloroform</td><td>VWR international</td><td>100777C</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>32221-2.5L</td><td></td></tr><tr><td>Trizol</td><td>Invitrogen</td><td>15596-018</td><td></td></tr><tr><td>RNeasy kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>SuperScript III Reverse Transcriptase</td><td>Invitrogen</td><td>18080-044</td><td></td></tr><tr><td>Immolase Taq</td><td>Bioline</td><td>BIO-21048</td><td></td></tr><tr><td>dNTP</td><td>Sigma-Aldrich</td><td>DNTP100A</td><td></td></tr><tr><td>Exonuclease 1</td><td>New England Biolabs</td><td>M0293S</td><td></td></tr><tr><td>Shrimp Alkaline Phosphatase</td><td>GE Healthcare</td><td>E70092Z</td><td></td></tr><tr><td>SpectroCLEAN resin</td><td>Sequenom</td><td>10053</td><td></td></tr><tr><td>MassEXTEND ddNTP/dNTP mix </td><td>Sequenom</td><td></td><td>Varies according to assay design</td></tr><tr><td>MassEXTEND thermosequenase </td><td>Sequenom</td><td>10052</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Chilled microcentrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>NanoDrop Spectrophotometer</td><td>Thermo Fisher Scientific, Inc.</td><td></td><td></td></tr><tr><td>SpectroPOINT nanolitre dispenser</td><td>Sequenom</td><td></td><td></td></tr><tr><td>SpectroREADER mass spectrometer</td><td>Sequenom</td><td></td><td></td></tr></tbody></table>

an allele-specific gene expression assay to test the functional basis of genetic associations

与常见的复杂特征显着的遗传协会的数量不断增加。然而，这些协会的大多数并没有被理解在分子水平上。介导的DNA变异的表型的影响的机制之一是，这已被证明特别是对复杂性状1相关的基因表达。 这种方法测试蜂窝上下文特定的DNA序列，基因表达的影响。其原理是相对丰度测量所产生的一个基因的两个等位基因，分析细胞进行与疾病（风险变种）2,3相关的DNA序列的一个副本成绩单。因此，这种方法使用的细胞应该满足了两个基本基因型要求:他们必须为DNA风险的变种和DNA分子标记，通常的编码基因多态性，基于他们的染色体的起源（图1）可以区分成绩单的杂合子。 DNA风险的变种和DNA标记不需要具有相同的等位基因频率，但阶段（haplotypic）的遗传标记的关系，需要理解。同样重要的是，选择感兴趣的基因的细胞类型，表达。此协议专指通过从成纤维细胞中提取核酸的过程，但该方法是同样适用于其他细胞类型，包括原代细胞。 从选定的细胞株DNA和RNA的提取和cDNA的生成。 DNA和cDNA进行了分析与设计目标编码DNA标记4，一个引物延伸实验。引物延伸实验进行使用MassARRAY（Sequenom公司）5平台，根据制造商的规格。引物延伸产品，然后分析了基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF/MS）时间。由于选定的标记是杂合子，他们将生成两个山峰上的MS情景。每个峰的面积是成正比的转录丰度和可与MassARRAY导电型测量仪软件功能生成一个等位基因的比例（等位基因1:等位基因2）测量计算。等位基因的cDNA获得的比例是使用从基因组DNA，其中等位基因的比例预计为1:1，以正确的技术构件规范化。与归一化的等位基因的比例显着不同的为1的标记表明，两个在同一个细胞的染色体产生的成绩单金额是不同的，这表明DNA与表型相关的变种基因表达的影响。应该用实验控制，以确认结果。

Watch this Scientific Journal Video about An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations at JoVE.com

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

遗传协会经常保持在功能级别上的原因不明。这种方法的目的是，通过分析细胞转录单核苷酸多态性杂合子基因表达，以评估的表型相关的遗传标记的影响。该技术可以精确测量，用MALDI - TOF质谱定量等位基因特异性引物延伸产品。

等位基因特异性基因表达检测，测试的遗传协会的功能基础

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

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JoVE Journal

Biology

22.8K 次观看.  University of Oxford. 遗传协会经常保持在功能级别上的原因不明。这种方法的目的是，通过分析细胞转录单核苷酸多态性杂合子基因表达，以评估的表型相关的遗传标记的影响。该技术可以精确测量，用MALDI - TOF质谱定量等位基因特异性引物延伸产品。

视频: An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

科研

遗传学

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

血栓烷 A2 受体基因 C924T 多态性的研究方法

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. While this has led to increased identification of likely pathogenic mutations associated with genetic syndromes, it has also dramatically increased the number of incidentally found genetic variants of unknown significance (VUS). Determining the clinical significance of these variants is a major challenge for both scientists and clinicians. An approach to assist in determining the likelihood of pathogenicity is signal-to-noise analysis at the protein sequence level. This protocol describes a method for amino acid-level signal-to-noise analysis that leverages variant frequency at each amino acid position of the protein with known protein topology to identify areas of the primary sequence with elevated likelihood of pathologic variation (relative to population &#34;background&#34; variation). This method can identify amino acid residue location &#34;hotspots&#34; of high pathologic signal, which can be used to refine the diagnostic weight of VUSs such as those identified by next generation genetic testing.

Amino acid-level signal-to-noise analysis determines the prevalence of genetic variation at a given amino acid position normalized to background genetic variation of a given population. This allows for identification of variant &#34;hotspots&#34; within a protein sequence (signal) that rises above the frequency of rare variants found in a population (noise).

遗传变异的氨基酸级信噪比分析确定变异致病性的可能性

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

癌症研究

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

应用生物标志物发现在前列腺癌:个别赫尔维位点表达微阵列鉴定

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

医学

Large-Scale Multi-Omics Genome-Wide Association Studies (Mo-GWAS): Guidelines for Sample Preparation and Normalization

Both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are widely used metabolomics approaches to detect and quantify hundreds of thousands of metabolite features. However, the application of these techniques to a large number of samples is subject to more complex interactions, particularly for genome-wide association studies (GWAS). This protocol describes an optimized metabolic workflow, which combines an efficient and fast sample preparation with the analysis of a large number of samples for legume crop species. This slightly modified extraction method was initially developed for the analysis of plant and animal tissues and is based on extraction in methyl tert-butyl ether: methanol solvent to allow the capture of polar and lipid metabolites. In addition, we provide a step-by-step guide for reducing analytical variations, which are essential for the high-throughput evaluation of metabolic variance in GWAS.

Large-Scale Multi-Omics Genome-Wide Association Studies Mo-GWAS: Guidelines for Sample Preparation and Normalization

In this protocol, we present an optimized workflow, which combines an efficient and fast sample preparation of many samples. In addition, we provide a step-by-step guide to reduce analytical variations for high-throughput evaluation of metabolic GWAS studies.

大规模多组学全基因组关联研究 （Mo-GWAS）:样品制备和归一化指南

Both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are widely used metabolomics approaches to detect ...

生物化学

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

生物学

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

定量检测，以研究蛋白质:DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

等位基因特异性基因表达检测，测试的遗传协会的功能基础

摘要

探索更多视频

此视频中的章节

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

血栓烷 A2 受体基因 C924T 多态性的研究方法

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

遗传变异的氨基酸级信噪比分析确定变异致病性的可能性

患者生物流体中肿瘤相关循环DNA的检测与监测

应用生物标志物发现在前列腺癌:个别赫尔维位点表达微阵列鉴定

大规模多组学全基因组关联研究（Mo-GWAS）:样品制备和归一化指南

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

定量检测，以研究蛋白质:DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Infinium检测的大型SNP基因分型应用

等位基因特异性基因表达检测，测试的遗传协会的功能基础

摘要

探索更多视频

此视频中的章节

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

血栓烷 A2 受体基因 C924T 多态性的研究方法

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

遗传变异的氨基酸级信噪比分析确定变异致病性的可能性

患者生物流体中肿瘤相关循环DNA的检测与监测

应用生物标志物发现在前列腺癌:个别赫尔维位点表达微阵列鉴定

大规模多组学全基因组关联研究 （Mo-GWAS）:样品制备和归一化指南

结合免疫荧光和三维保存的间期核荧光原位杂交DNA研究在3D核组织的变化

定量检测，以研究蛋白质:DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Infinium检测的大型SNP基因分型应用

大规模多组学全基因组关联研究（Mo-GWAS）:样品制备和归一化指南