亚硫酸氢转换协议下面介绍一个标准协议为2微克的基因组DNA的转换。数额较小或较大的基因组DNA（100 PG - 200微克），也可以成功治疗的亚硫酸氢。这些反应条件，结果在完成转换几乎所有的目标DNA 序列 3（99.5-99.7％） 。 1。 DNA的制备准备通过孵化与亚硫酸氢DNA裂解液总量在37 ° C 注意：在1小时18μL（2微克吨 RNA的，280毫微克/μL蛋白酶K，1％SDS）的基因组DNA 样本：这是重要的实现最大的亚硫酸氢转换，尤其是从临床标本中，仍然有可能与DNA相关的蛋白质分离的DNA。 2。 DNA变性<ol><li>在20μL体积的变性2微克的DNA，2μL新鲜配制的3M NaOH溶液中加入一个最终的Concentration为0.3m。 </li><li>样品在37 ° C的水浴中15分钟，随后在90℃孵育，在一个加热块2分钟。立即置于冰上5分钟管。 </li><li>离心管于4 ° 10小号在10000 克 ，以确保DNA在试管底部。 </li></ol> 3。亚硫酸氢脱氨磺化与水解脱氨<ol><li> 10毫米醌和饱和焦亚硫酸钠pH值5.0（7.6克钠2 S 2 O 5的10 M NaOH与464μL，15毫升水）准备新鲜的解决方案。饱和亚硫酸氢钠溶液是通过轻轻颠倒试剂/ H 2 O混合，以最小的混合和曝气。如果有必要，10米的NaOH调整pH值，充分溶解的焦亚硫酸钠注意：由于它是一个饱和溶液，小疙瘩可能仍然不溶。 </li><li>新增208饱和焦亚硫酸钠和12μL变性的DNA（20μL）为10mm醌在240μL至终浓度为2.31M bisulphite/0.5mM醌，pH值5.0的最终体积，μL。轻轻混合所有试剂和离心10秒，以确保所有的水滴在试管底部。 </li><li>覆盖200μL矿物油的样品。在55 ° C水浴中，为4-16小时孵育样本。亚硫酸氢处理的长度是要转换的DNA的数量和质量上的依赖。对于质量较差或降解DNA的DNA，限制孵化时间为4 小时注：重要的是，亚硫酸氢转换发生在黑暗的地方，以避免氧化，所以如果是没有可能的包装铝箔管到孵化前。 </li><li>在适当的孵化时间结束，在离心旋转管简单，以确保所有的液体在试管底部。 </li><li>恢复的亚硫酸氢处理的DNA仔细移液器DNA溶液从试管底部没有采取任何矿物油，从下一层油。 </li></ol>脱盐<ol start="5"><li>移除任何免费的亚硫酸氢离子通过一个脱盐柱，并在50μL（MQH 2 O）的Milli - Q水洗脱。根据基因组DNA的数量和质量上和它是如何准备的，可用于不同脱盐列。我们经常使用Promega公司向导清理列，这些列适合去除SDS编制的DNA转换之前使用。 </li></ol>碱Desulphonation <ol start="6"><li>亚硫酸氢加合物从尿嘧啶环desulphonation。 Desulphonate 5.5μL新鲜配制的3M NaOH溶液加入到终浓度为0.3m，然后在37 ° C，持续15分钟的孵育样品。 </li><li>短暂离心，并添加1μL 的 T RNA（10毫克/毫升） 。 </li> &lt;李&gt;中增加33μL醋酸铵（NH 4 O AC），pH值7.0的3M公司的终浓度的解决方案。 </li><li>乙醇沉淀，加入冰冷的100％的乙醇330μL的DNA，拌匀反转。 -20μC离开1小时至过夜。在15分钟的14000克离心4 ° C 。清除所有痕迹上清，空气干燥〜20分钟沉淀DNA。 </li><li>暂停0.1 TE（10毫米的Tris - HCl，0.1mm的EDTA，pH值8.0）或H 2 O 50μL/微克DNA沉淀在室温下放置约2小时。偶尔涡管，以确保DNA溶解，快速离心机。 </li><li>立即用于PCR扩增，或在-20 ° C存储的DNA的数量和质量而定为1-10年。 </li></ol> 4。 PCR扩增引物设计<ol><li> PCR亚硫酸氢转换特异引物的有效设计crucia为确保高效，公正完全转化的DNA扩增升发生。以下准则用于帮助引物设计。 </li></ol>引物设计准则<img src="files/ftp_upload/3170/3170list1.jpg" /> Thermocyling <ol start="2"><li>要测试比例的甲基化和甲基化DNA的PCR扩增，使用一个甲基/甲基化50:50全亚硫酸氢转换控制样品和扩增的亚硫酸氢转换特定引物下，优化PCR反应条件（图3）。 50:50的甲基化：甲基化控制，使用体外 SSSI甲基化的DNA，要么全基因组扩增（WGA）的DNA或全血DNA。请注意，有些基因会自然血液中的甲基化，因此在这些情况下，最好是只使用WGA“甲基化”控制DNA。 </li><li>准备PCR扩增反应混合物在100＆MU;升等份，含2μL亚硫酸氢转换的基因组DNA，200微米的dNTP的，1μM引物，1.5毫米，50 mM的氯化钾，10毫米的Tris - HCl，pH值8.3 和0.15μLTaq聚合酶MgCl 2的。 </li><li>在温度梯度热循环仪，设置在一个渐变的运行反应+ / - 3 ° C，从预测的Tm的引物在10个管</li></ol><img alt="表1" src="/files/ftp_upload/3170/3170table1.jpg" /><ol start="5"><li>为了测试，甲基化和甲基化扩增产物已按比例放大，扩增被消化信息的限制性内切酶， 如Taq酶1（TCGA），将消化甲基化的DNA，但不会消化甲基化的DNA限制性内切酶网站时改变后的亚硫酸氢转换到TTGA。消化的程度，可以通过琼脂糖凝胶电泳（图3A）可视化。另外，SYBRGreen（1:1000稀释液0.2）可添加到PCR反应和甲基化的程度可以评估在实时PCR热循环仪（图3B）进行热分解图。 </li><li>可能需要进行调整，以提高PCR的效率或比例，以确保甲基化和甲基化PCR片段的PCR扩增PCR反应混合物包括MgCl 2的浓度和热循环条件。 </li><li>一旦PCR条件进行了优化，PCR扩增，可以进行亚硫酸氢转换样本注：一旦以旧换新优化温度梯度PCR需要不能使用。 </li></ol> 5。代表性的成果： 亚硫酸氢盐PCR扩增优化的一个例子是如图3所示。优化PCR扩增条件应放大比例和不带偏见的甲基化和甲基化的扩增产物。图3A显示琼脂糖凝胶温度梯度PCR扩增了50％的DNA甲基化和甲基化混合物轮廓。钍经治疗后已发送PCR扩增，限制性内切酶，Taq酶1（TCGA），消化甲基化（M）的DNA，但不会消化甲基化（U）的DNA限制性内切酶的网站是由亚硫酸氢盐转化为TTGA改变。切割和未切割的PCR产物等量预计，如果DNA甲基化和甲基化比例放大。可以看出，这种凝胶，非偏置放大的最佳thermocyling条件是在56.1 ° C（T）。也可以完成转换的亚硫酸氢DNA进行分析与HPA三（CCGG）胞嘧啶网站，如特定的酶消化。 帕安 III只会消化，如果转换失败，限制站点将保持不变。如果在转换完成的限制，现场将被转换TCGG或TTGG根据的DNA甲基化状态。完整的亚硫酸氢DNA的转换，可以看出，在图3A（高）。 图3b显示实时解离积也可以用来作为一种工具，以确定是否有是基于不同的分子离解时的温度的任何扩音偏置。在这个数字中可以看出的PCR扩增甲基化（M）和甲基化（U）的DNA中的比例从50％的DNA甲基化和甲基化混合比控制完全甲基化和甲基化的DNA扩增，其中游离在82.1μC 78.9μC分别。 经过thermocyling和反应条件的优化亚硫酸氢处理的样品可以与钢绞线的具体和亚硫酸氢在单个或半巢式PCR反应的特异性引物扩增。由此产生的PCR片段可以可视化琼脂糖凝胶电泳和直接（图4A）或通过克隆和测序测序。克隆和测序单个分子的甲基化状态后，可以在亚硫酸氢地图（图4B），表格，可视化的异质性甲基化。 高通量定量甲基化分析，可以预制使用Sequenom公司EpiTYPER方法利用技术等。使用这种方法，亚硫酸氢转换的DNA与亚硫酸氢特异性PCR引物扩增和proprietry裂解过程。产生的碎片是依赖于甲基化的胞嘧啶（图5A）的存在与特定的频谱用MALDI - TOF质谱定量分析。样本中的甲基化比率的摘要，然后可以推断警句（图5B）或甲基化曲线（图5C）的形式。 <img alt="图1" src="/files/ftp_upload/3170/3170fig1.jpg" /> 图1。在正常细胞的甲基化的CpG原理图，促进相关的主要是甲基化CpG岛（灰色），而稀疏，一般甲基化（红色）的CpG位点基因机构内。在右侧面板扩展的分子结构茜在个别CpG位点的DNA，并显示在5碳的胞嘧啶甲基分子的甲基化。 <img alt="图2" src="/files/ftp_upload/3170/3170fig2.jpg" /> 图2。化学转化原理的DNA甲基化分析包括四个主要阶段，如下所示;变性，亚硫酸氢盐转化，PCR扩增和分析。在右侧面板中，修改胞嘧啶分子，亚硫酸氢转换过程中出现的描绘。 <img alt="图3" src="/files/ftp_upload/3170/3170fig3.jpg" /> 图3。优化PCR扩增：A.琼脂糖凝胶电泳，温度梯度PCR扩增了50％的DNA甲基化和甲基化混合物轮廓。要么Taq1（T）或HpaIII（H）分别消化的DNA甲基化和非亚硫酸氢转换的DNA片段已被消化。 B.热解离曲线分析显示了一个方程L的DNA甲基化和甲基化（红线50/50混合）的比例相比，完全甲基化的控制放大（粉红线，M）和甲基化DNA（绿线，U）为82.1游离于° C和78.9 ° C分别。 <img alt="图4a" src="/files/ftp_upload/3170/3170fig4a.jpg" /> <img alt="图4b" src="/files/ftp_upload/3170/3170fig4b.jpg" /> 图4。直接测序的例子和亚硫酸氢地图 。从三个不同的细胞系，以黄色突出显示的CpG位点的序列跟踪。细胞系X显示100％，在所有3个CpG位点的甲基化，而细胞系的Y和Z显示的甲基化程度不同，重叠G / A的信号。 B.代表亚硫酸氢地图显示在个别CpG位点甲基化的密度（红点），由单个克隆直接测序确定。 <img alt="图5a" src="/files/ftp_upload/3170/3170fig5a.jpg" /> 图5。高通量DNA甲基化分析Sequenom公司。A。频谱视图使用Sequenom公司epiTYPER技术。 DNA片段显示特定的光谱，DNA甲基化目前量而定。 B.警句汇总在四个不同的细胞系的每个CpG位点的DNA甲基化的比例。 C.甲基剧情梗概来自Sequenom公司警句。

<ol>
	<li>Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomic+sequencing+protocol+that+yields+a+positive+display+of+5-methylcytosine+residues+in+individual+DNA+strands.">A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.</a> Proc. Natl. Acad. Sci. U. S. A. 89, 1827-1831 (1992).</li><li>Clark, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+mapping+of+methylated+cytosines.">High sensitivity mapping of methylated cytosines.</a> Nucleic. Acids. Res. 22, 2990-2997 (1994).</li><li>Grunau, C., Clark, S. J., Rosenthal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisulfite+genomic+sequencing:+systematic+investigation+of+critical+experimental+parameters.">Bisulfite genomic sequencing: systematic investigation of critical experimental parameters.</a> Nucleic. Acids. Res. 29, E65-E65 (2001).</li><li>Clark, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation:+bisulphite+modification+and+analysis.">DNA methylation: bisulphite modification and analysis.</a> Nat. Protoc. 1, 2353-2364 (2006).</li></ol>

没有利益冲突的声明。

亚硫酸氢转换的DNA基因组测序的DNA甲基化分析是一个完善的和灵活的方法，便于识别和量化DNA甲基化的单核苷酸分辨率。然而，亚硫酸氢转换和随后的分析依赖的DNA已经完全符合每一个甲基化的胞嘧啶被deaminated尿嘧啶和甲基化的胞嘧啶只，其余未反应转换的前提。如果转换是不完整的，可能会出现问题分析，因为归正的甲基化的胞嘧啶甲基化的胞嘧啶可能会错误地解释。亚硫酸氢转换可以优化在不同阶段，最大限度地转化率。对于DNA全部转为首先必须是单链，使胞嘧啶残基暴露的亚硫酸氢离子。第一步，DNA变性，是至关重要的，可以是不完整的转换的源，如果DNA是不完全变性。为了确保的DNA完全变性是很重要的反应参数，包括清除所有相关的蛋白质，适当的盐分浓度，孵化温度和时间都适合保持在单链构象的DNA。重要的是用新鲜的3M NaOH和，以便有足够的孵化时间。如果DNA是抗变性，一些协议的修改，包括孵化时间延长至30分钟或碎片的DNA变性前，可促进DNA变性。此外，单链DNA（ssDNA的）可以重新退火双双链DNA（dsDNA）的过程中的亚硫酸氢转换反应。开展定期或不断在亚硫酸氢反应在较高的温度（90-95℃）的反应，可以帮助维修单链DNA。然而，重要的是要知道，在这些较高的温度大大加速DNA的降解。各种试剂也可以添加到扰乱日重新退火发送的DNA链，如尿素。 DNA和其质量的浓度也影响亚硫酸氢反应的效率和最终的PCR扩增产量。 DNA降解的亚硫酸氢转换协议的限制。化学处理的DNA引入单链DNA的各种链断裂和完整的亚硫酸氢转换，包括亚硫酸氢浓度高，潜伏期长的时间可以加速DNA的降解所需的一些苛刻的条件。标准反应条件下，DNA降解不但一个共同的问题，如果协议的修改出台，如难治性转换的序列，如FFPE样品的质量较差或退化的DNA模板，然后DNA降解可以成为一个显著的限制。 福尔马林固定，石蜡包埋（FFPE）和退化的DNA样本，可以有效地亚硫酸氢使用此协议的转换和放大，但是建议修改，以确保DNA是不是进一步退化。 如 t RNA的载体RNA的建议使用。也糖原时，可以添加为载体DNA浓度低（&lt;200吴）。因为从FFPE组织中分离出的DNA已经分散和进一步分散在脱氨的地方，必须小心，以尽量减少进一步恶化。不要任何进一步的片段DNA变性和限制前孵育时间为4个小时的亚硫酸氢反应。设计扩增不超过300个基点，由于分散的DNA。 另一个因素可以影响PCR扩增甲基化胞嘧啶的亚硫酸氢转换，降低DNA模板的复杂性。在4.1节中，增加引物的长度和巢式PCR所述以下的引物设计的协议都可以帮助增加特异性和PCR扩增产量。 最后，由于DNA模板改变在bisulphITE转换，放大偏置可关注。确保检查比例的甲基化和甲基化的模板的PCR扩增与控制50/50 / ü DNA混合，如在协议中所述，见图3。此外，还要确保只有转换模板的扩增，是发生使用HpaIII限制性内切酶和凝胶电泳（图3）。 PCR反应条件，可能需要不同的温度和/或镁离子浓度或延长延伸时间进行了优化。有些模板出现问题尤其严重，引物的位置可能需要调整或简并引物，可能需要使用。 下一代测序技术正在迅速发展，实现了类似的决议，以亚硫酸氢的基因组测序，第三代单分子测序的潜力，让小学的DNA甲基化的分析，而不需要为亚硫酸氢盐测序。然而，广泛的可用性和规格ificity这些技术仍然尚需时日。亚硫酸氢盐基因组测序中的甲基化分析的黄金标准，是一个强大的协议。该方法已取得进展，已解决共同的问题和局限性和应用程序从个人的基因分析扩大到高吞吐量和克拉克等人所描述的整个基因组 的分析，2006年4。表1列出故障排除指南和其他建议。这是建议的最佳方法是最初遵循的标准协议，只介绍修改，如果在出现问题时。 <img src="/files/ftp_upload/3170/3170table2.jpg" alt="表2" /> 表1故障排除准则。

<ol><li>焦亚硫酸钠（阿贾克斯精细化工） </li><li>对苯二酚（默克） </li><li>向导的DNA清理系统（Promega公司） </li></ol>

dna methylation: bisulphite modification and analysis

表观遗传学描述基因功能的遗传发生的变化，独立的DNA序列。后生基因表达调控的分子基础是复杂的，但本质上涉及到的DNA本身或与DNA联营蛋白质的修改。在哺乳动物的基因组DNA表观遗传修饰的主要是甲基化的胞嘧啶核苷酸（5 - MEC）。 DNA甲基化的基因表达机械提供指令时应该表达的基因。在哺乳动物DNA甲基化的主要目标序列的CpG - 5&#39; - 3&#39;二核苷酸（图1）。 CpG二核苷酸并非均匀地分布在整个基因组，但都集中在重复的基因组序列的CpG的，通常与基因启动子（图1）相关的“孤岛”的区域。 DNA甲基化模式是建立在发展初期，在组织特异性分化调制，并在包括癌症在内的许多疾病状态的干扰。到Understand的生物学作用的DNA甲基化，其作用在人类疾病中，精确，高效和可重复性的方法，检测和量化个人5 MECS。 这为亚硫酸氢转换的协议是“黄金标准”DNA甲基化分析，并便于识别和量化的DNA甲基化的单核苷酸分辨率。亚硫酸氢钠的胞嘧啶脱氨的化学包括三个步骤（图2）。 （1）磺化：除了胞嘧啶（2）水解脱氨5-6双键：对胞嘧啶亚硫酸氢产生的衍生给予尿嘧啶亚硫酸氢衍生物（3）碱Desulphonation水解脱氨的亚硫酸氢去除磺酸经碱处理组，给尿嘧啶。亚硫酸氢优先deaminates胞嘧啶尿嘧啶在单链DNA，而5 - MEC，是难治性亚硫酸氢介导的脱氨。 PCR扩增后，尿嘧啶是放大胸腺嘧啶，而5 - MEC的残留物仍然为胞嘧啶，允许由来自甲基化CpGs的胞嘧啶的“C”与胸腺嘧啶“T”的残留物的存在，在测序来加以区分甲基化CpGs。 DNA的亚硫酸氢盐转化的修改是一个完善的协议，可利用DNA甲基化分析的方法很多。由于5 - MEC检测亚硫酸氢转换是首次展示由Frommer 等 1和克拉克等。2，围绕基因组DNA帐户的亚硫酸氢转化为大多数对DNA甲基化的新数据为基础的方法。可利用不同的方法后PCR分析，取决于特异性和甲基化的决议要求程度。克隆和测序仍是最容易获得的方法，可以给整个DNA分子中的甲基化单核苷酸分辨率。

Watch this Scientific Journal Video about DNA Methylation: Bisulphite Modification and Analysis at JoVE.com

DNA Methylation: Bisulphite Modification and Analysis

DNA甲基化分析的黄金标准是亚硫酸氢转换的DNA基因组测序。 5 - 甲基胞嘧啶（5 - MEC）亚硫酸氢在酸性条件下脱氨相比，这种方法需要利用胞嘧啶的敏感性增加。后PCR扩增目标基因组DNA甲基化的胞嘧啶甲基化的胞嘧啶可以区分。

DNA甲基化：亚硫酸氢修改和分析

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

dna-methylation-bisulphite-modification-and-analysis

Research

JoVE Journal

Biology

104.1K 次观看.  Garvan Institute of Medical Research. DNA甲基化分析的黄金标准是亚硫酸氢转换的DNA基因组测序。 5 - 甲基胞嘧啶（5 - MEC）亚硫酸氢在酸性条件下脱氨相比，这种方法需要利用胞嘧啶的敏感性增加。后PCR扩增目标基因组DNA甲基化的胞嘧啶甲基化的胞嘧啶可以区分。

视频: DNA Methylation: Bisulphite Modification and Analysis

PDMS Device Fabrication and Surface Modification

PDMS的设备制造和表面改性

科研

生物学

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

印记基因的DNA甲基化的测定拟南芥胚乳

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

基因改造和重组唾液的腺体器官培养

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

样机小型化流式细胞仪及配套的混合微流控技术降低重力环境硬件演示

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

在体外甲基化分析研究蛋白质精氨酸甲基化

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

增强的减少表示亚硫酸氢盐测序的DNA甲基化的评估，在碱基对决议

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.