We thank Timothy F. Sharbel (IPK Gatersleben) for providing Boechera divaricarpa seeds and Sharon Kessler (University of Oklahoma) for critical reading and proofreading. Work on cell type-specific transcriptome analyses to study gametophyte development and apomixis in UG&#180;s laboratory is supported by the University of Z&#252;rich, by a fellowship of the "Deutsche Forschungsgemeinschaft" and the Marie Curie project IDEAGENA to AS, by grants from the "Staatssekretariat f&#252;r Bildung und Forschung" in the framework of COST action FA0903 (to UG and AS) and the Swiss National Foundation (to UG).

注:本协议描述的组织准备，激光辅助显微解剖，并转录谱RNA提取。始终使用手套整个协议的所有步骤。研究和考虑所使用的每种化学品的安全说明。特别是，要记住，二甲苯是有害的，并且可以穿透手套和甲醇是有毒的。对于所使用的所有工具，请相应参考用户手册。 1.玻璃制品及其他设备拆除RNase活性<ol><li>在使用之前，包裹在铝箔任何玻璃器皿在180℃烘烤〜8小时，以确保无RNA酶的质量之前。 </li><li>治疗任何金属表面（ 例如，镊子），以及在工作台上，并用适当的RNA酶去污溶液加热板。此后，洗净表面用无RNase水以去除净化解决方案。接着，用70％乙醇进一步清洗的表面上。 </li><li>使用所有纤溶酶抽动消耗品（如管）全新的无任何预处理。如果可能，使用塑料耗材经过认证可以自由地RNA酶。 </li></ol> 2.组织固定<ol><li>在冰上新鲜15ml试管:;:准备10毫升农民的固定液（乙酸3 V / V乙醇1）。 （总是准备使用前新鲜农民的固定液）。作为一种替代方案中，非交 ​​联固定剂乙醇:乙酸（5:1;体积/体积）可以使用18。 </li><li>用细镊子在感兴趣的发育阶段收集芽。立即淹没在冰冷的固定液芽。使用10毫升固定液为固定〜来自拟南芥或Boechera 20芽或花注意 :当使用具有较大的花的植物品种时，建议解剖用刀片的花之前定影产生更小的碎片。 <ol><li>为了获得成熟的配子体，阉割了2 - 3大花inflor芽escence在收获之前2天。 </li></ol></li><li>填用冰冷的干燥器中的底部。打开装有样本， 例如，花管，并将它们放置在冰上，通过直接将它们放入冰的方式，它们不能翻倒或通过使用适当的保持器。真空渗透了以释放真空之前15分钟。 <ol><li>重复真空渗入一次15分钟。 </li></ol></li><li>释放在冰上真空和存储过夜（〜12小时）。覆盖冰桶用盖或铝箔，并在4℃的室温或冰箱铲斗存储，以防止从解冻冰。注意:不建议延长固定时间。 </li></ol> 3.组织包埋，薄切片，并在幻灯片安装了LAM <ol><li>组织包埋。 <ol><li>交换固定液用纯乙醇和无RNA酶的水配制70％的乙醇。留在冰样品，直到进一步的处理。启动EMBE样品的dding同一天。在任何情况下，组织加工机可用，进行手工嵌入其他地方所描述9,11和继续执行步骤3.1.11。 </li><li>轻轻的样品用镊子转移到组织包埋盒中。关紧磁带。关键的一步:在此步骤避免样品甚至局部干燥。避免与镊子样的挤压。 注意:要标记铅笔应使用的磁带盒，最好由磁带盒的倾斜表面上书写。 </li><li>存储装入70％的乙醇的芽或花，直到所有材料被转移到盒和嵌入机器可装载的包埋盒。为了这个目的，可使用填充有70％的乙醇的玻璃染色槽。 </li><li>从机器的蒸馏除去组织处理器的筐和嵌入盒具有面向上方的倾斜面，并在相同的方向转移到篮。 CRI蒂卡尔步骤:快速执行此，以防止组织任何干燥。如果同时处理许多包埋盒，将篮子到装载样品之前填充有70％的乙醇的适当无RNA酶的容器中。 </li><li>放在篮子盖子，把篮子放回反驳。关闭反驳。 注:组织处理器自动脱水样品，改变溶剂二甲苯，并不会融化的石蜡渗透。通常，这是过夜进行。 </li><li>启动组织处理器的程序。推荐的标准设置为1小时70％乙醇，三次1小时90％EtOH中，三次1小时100％EtOH中，两次1小时100％二甲苯，一时间1小时15分钟100％二甲苯在室温下，随后的渗透用石蜡1小时，并在56℃下11一次3小时两次。 注:为了确保阻塞站愿与融化的蜡，开关在机器上几个小时开始之前或使用定时器功能选择的近似起始时间。 注意:如果该组织不能被随后立即在56℃下进行处理，在石蜡更长的温育时间在56℃下是可能的，而不是3小时。参阅仪器的用户手册。通常情况下，直到"空反驳"命令批准了组织中的磁带会自动停留在熔化的蜡。如果在启动组织处理器当蜡不熔化，蒸馏填充有70％的乙醇和程序保持搁置，直到准备好开始。 </li><li>空蒸馏，并立即将样品在56℃的阻挡站转移到石蜡浴。从篮下取下盖子，盖上了井盖，封锁站的石蜡浴。 </li><li>阻挡台的表面上堆一样多的新鲜平衡塔板加热至56℃，因为有在篮子盒。 注:耐乙醇永久性标记可使用d，来标记平衡托盘的底部。不推荐使用耐丙酮永久性标记。 </li><li>在56℃下使用的阻塞台，充满熔融石蜡一个预热新鲜塑料平衡托盘。抬起石蜡浴的盖，只在一个时间拾起一个盒和使用一对镊子的在平衡盘在56℃下从所述盒转移样品到液体石蜡。 关键的一步:避免周围的样品，同时通过快速处理工作卡带蜡中的任何硬化。 <ol><li>根据使用的制剂针蜡的硬化前的需要的所有样本的位置。典型地，几个花卉布置在平衡盘在每个方向分别隔开约1厘米。 </li><li>重复步骤3.1.9，直到所有样品转移。 </li></ol></li><li>放置筐回组织处理器并使用适当的程序（请参考用户手册）清洁反驳。 </li><li>关闭组织的处理器和拦截站。步骤3.1.13继续。 </li><li>万一没有组织浸润机和没有嵌入站是可用的，可使用加热烘箱中在56℃以熔化石蜡。在板凳上，填补了塑料托盘的平衡熔化的蜡，样品转移到蜡，并用准备针定位样品。 </li><li>允许石蜡在室温下为〜1变硬 - 在4℃下储存样品之前3小时。样品随后可以新鲜处理或贮存长达几个月。 </li></ol></li><li>薄切片和幻灯片的LAM的制备。 <ol><li>一盏灯表从下面照亮蜡块，删除与从周围的塑料托盘样本的石蜡。解剖出包含组织中的平方蜡块， 例如 ，一芽一叶或花，用无RNA酶的刀片。为此目的，总是定向朝向蜡块顶部的样本（也见<str翁&gt;图1）。 <ol><li>准备应在同一时间被用于LAM所有样品的蜡块。 </li></ol></li><li>安装块来处理嵌入填充有硬化石蜡盒:使用乙醇灯熔体上的适当刮刀的前端的小片或石蜡的液滴，并使用热蜡固定在载体上的块（​​带有包埋组织在上面）。 <ol><li>使蜡在4℃下固化至少20分钟。 </li></ol></li><li>删除多余的蜡与周围的光台刀片样品。请一定要保持一个正方形表面平行边（ 见图1）。 </li><li>设置在加热板至42℃。 </li><li>安全地装入样本以切片机和制备6 - 10微米厚的切片。指制造商的说明使用切片机。注:虽然较薄部分通常会给出更好的结构的分辨率，更大的解释第1条RNA的量ř质量可以与厚的部分来获得。对于Boechera的细胞成熟配子体，我们使用7微米为默认值。移动相当缓慢的样品在切片刀通常会导致更好的组织学。 </li><li> 15cm宽黑色纸板在底部LAM幻灯片定位在他们面前成为一个塑料盒 - 始终在10左右的长度转移石蜡丝带。 </li><li>如果可能的话，预选择包含细胞或组织类型的兴趣， 例如，胚珠石蜡条的部分，下一个解剖-范围并丢弃其余部分。 </li></ol></li><li>幻灯片为LAM的制备。 <ol><li>放置的金属框架的滑动所需要的量（一般为5 - 10）上的加热板顶部的平的表面。 </li><li>吸管〜1 - 2毫升无RNA酶的水的上滑动的塑料部分。使用刀片，切石蜡带约4小品 - 5厘米足够长，以跨越塑钢窗。 ü瑟镊子和制备针对理想地放置相互平行的石蜡条上滑动的塑料窗户。小心地提起滑盖一端，除去水，并把幻灯片回来。 <ol><li>或者，将加热板下的化学罩。适用的甲醇的体积小到滑动刚好足以润湿表面的塑料部分（这可能会导致该幻灯片的一个轻微的波纹）。广场上的幻灯片石蜡条。 注意:对于毒性的原因，避免使用甲醇如果可能的话。然而，在这个步骤中使用的甲醇中确实改善了RNA的质量的情况下，由安装在水达到的质量是不够的。这是值得测试这些替代品的一个新项目的启动，为实现RNA的质量与被调查细胞类型和品种而异。 </li></ol></li><li>过夜在加热板上干燥载玻片在42℃〜12小时。覆盖与加热板塑料盖不触及幻灯片。确保该塑料盖不会阻止空气交换。到这一目的，盖子可以放在吸管盒或类似被提升。 <ol><li>可替代地，缩短干燥时间，以至少两小时或直至组织出现完全干燥。时间从切片到收集的减少可能会提高RNA的质量。 </li></ol></li><li>在化学罩，脱腊幻灯片2次二甲苯中10分钟，使用适当的幻灯片持有人。确保完全淹没每张幻灯片的塑料部分，但仅通过触摸保持干燥金属框架的更广泛的结束，让去除幻灯片。 </li><li>允许的幻灯片之前，林化工引擎盖下干燥〜10分钟。 注意:幻灯片不立即处理可以在42℃下被放置回加热板与组织朝上长达几个小时。这将防止湿气影响RNA的质量，直到进一步的处理。 </li></ol></li></ol> 4.激光辅助显微切割（LAM） 注意:LAM的程序将与所使用的仪器而变化。这个协议的某些细节适于特定仪器和收集上的盖利用静电力的样品的技术。另外，细节可能甚至不同版本的软件的之间变化。请参照制造商的说明书和用户手册进行了详细的描述和对仪器和软件的具体说明。 <ol><li>开关设备上的LAM（电脑，显微镜，激光控制箱）。 </li><li>启动程序转向显微镜和激光。 </li><li>通过按下控制箱上的相应的按键开关上的激光。 </li><li>放置一个盖子（0.5毫升，而不扩散器）插入盖支架和它在仪器的位置。 </li><li>支持与显微镜载玻片上LAM幻灯片。确保与滑动的平坦表面连接组织面临的搏命。 </li><li>将在下面的载玻片仪器的幻灯片。 </li><li>选择4X目标。在节目中，选择要盖移动到"向上"位置，并继续使幻灯片的扫描。 </li><li>搜索通过幻灯片，并确定感兴趣的结构。确定并保存在幻灯片上它们的位置，以便能够移动回到用于切割步骤的确切位置。 </li><li>选择适当的目标（ 如 40X 60X或）。 </li><li>如果需要的话，调整激光速度，焦点和激光功率，并且还通过参照器械的用户手动校准阶段移动。一旦一个帐户设置为感兴趣的特定组织中，设置可以被重复使用。 </li><li>验证通过按程序的"激光照射"按钮，理想地在膜的无组织的一部分的单杆激光位置。 <ol><li>如果需要，按照日校准激光位置Ë制造商的仪器说明书。 </li></ol></li><li>通过移动一个明显的结构，以在监视器上的软件窗口的全部四个角，保持按下鼠标按钮验证目标的校准。检查相对于明显的结构的手的位置是在所有四个角相同。否则，校准后的软件手册中的目标。 </li><li>凭借在"向下"的位置盖，用"手钢笔"工具标记细胞类型或感兴趣的组织， 例如，卵细胞的边界。按&#39;一刀切&#39;解剖与激光感兴趣的细胞。注意:如果单元格的边框不是一次完全解剖通常情况下，切片需要重复。在这种情况下，建议设置软件自动执行部分作为标准的两个重复。 </li><li>抬起盖并移动到与感兴趣的结构中下一个储存位置。重复步骤4.13该过程，直到从一个幻灯片的所有感兴趣的细胞被收集到解剖下一个单元的部分。确保帽被定位的方式，新的第坚持在盖的空位置。 注:建议以分离较大块的组织（ 例如，全花或部分段）从单细胞段上的新鲜帽隔离之后的每个滑动。这些可用于RNA的质量控制。 </li><li>删除幻灯片，重复步骤4.4继续下一个幻灯片 - 4.9和4.13至4.14。 </li><li>重复步骤4.15和有关步骤，直到细胞类型的所有幻灯片的兴趣已经分离。注意:不建议收获超过〜4个长 - 在同一个帽5小时。 </li><li>目视检查瓶盖表面LAM后排除样品的非特异性污染。同时非解剖组织与该箔的保护，灰尘可以粘到表面因静电附着力。 <ol><li> ŧØ目视检查盖，取出从仪器的幻灯片。将罩架在"向下"的位置，并用4X客观检查面。 </li><li>万一尘土小块是可见的，具有小的（2微升）枪头删除它们。可替代地，安装在滑动背面上的仪器，放置在幻灯片（无组织）的自由部分的帽，以及使用该激光器完全破坏污染。 </li></ol></li><li>关闭帽和冷冻干燥的部分在-80°C，直到进一步使用。如果需要的话，可将样品存储长达数周。但是，建议在这一步很快进行。 </li></ol> 5. RNA分离及质量控制注意:RNA可通过适合于少量的RNA的任何方法来分离。在这个协议中的使用为少量RNA的指定的RNA分离试剂盒的说明。按照制造商的说明。 <ol><li>使用管与连接到样品盖子直接或从-80℃取出后。 <ol><li>打开管并小心地吸取11微升提取缓冲液使用过滤嘴每个管的壁。更改样本之间的秘诀。 </li><li>盖上盖子并摇动提取缓冲液至管的盖子。 </li><li>放置与帽管向下在预热至42℃的加热烘箱中。孵育按照制造商的指示30分钟。 </li></ol></li><li>遵循制造商的说明列制备，并在管的底部收集提取物。 <ol><li>如果从一个以上的帽材料需要被组合成一个样本，进行与步骤5.1.1 - 5.1.2单独为每个管/盖。 </li><li>此后，汇集的萃取一个样品在一个管并加入EtOH中的等效量的事先测量汇集提取物用移液管量（见生产商的说明）。 </li><li>确保的Et的量OH加入前装入柱等于合并提取物的体积。 </li><li>用EtOH RNA沉淀之后，快速进行下列制造商的说明中列的负载。继续与100 XG离心步骤的协议。 </li><li>后的各收集管中的内容已通过该柱，进行16,000 xg离心离心步骤，持续30秒，以除去流过下列制造商的说明。 </li><li>具有下列制造商的说明提取RNA和纯化继续。 </li><li>洗脱缓冲液的柱的装载后允许柱孵育1分钟。继续到管放置按照制造商的说明中的说明离心机。注意:在洗脱前先100 xg离心1分钟的离心分离步骤如在协议指示可以增加洗脱的效率。 </li></ol></li><li>从续提取RNAROLS（较大的组织切片）进行RNA质量控制以下步骤5.1本协议5.2.7。 </li><li>用于RNA质量控制负载1μl的使用RNA微微芯片按照制造商的说明，在生物分析仪各对照样品的未稀释的总RNA。 注意:提取的RNA可用于线性放大和使用微阵列或RNA测序7,11,13,14转录组分析。理想情况下，质量控制应指示的RNA完整性数（RIN）的至少7.一些供应商提供用于扩增合适的试剂盒，合适的例子是在9给出。 </li></ol>

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W., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+germline+formation:+molecular+insights+define+common+concepts+and+illustrate+developmental+flexibility+in+apomictic+and+sexual+reproduction.">Plant germline formation: molecular insights define common concepts and illustrate developmental flexibility in apomictic and sexual reproduction.</a> Development. 142, 229-241 (2015).</li><li>Schmidt, A., 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=Apomictic+and+sexual+germline+development+differ+with+respect+to+cell+cycle,+transcriptional,+hormonal+and+epigenetic+regulation.">Apomictic and sexual germline development differ with respect to cell cycle, transcriptional, hormonal and epigenetic regulation.</a> PLOS GENET. 10, e1004476 (2014).</li><li>Okada, T., 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=Enlarging+cells+initiating+apomixis+in+Hieractium+praealtum+transition+to+an+embryo+sac+program+prior+to+entering+mitosis.">Enlarging cells initiating apomixis in Hieractium praealtum transition to an embryo sac program prior to entering mitosis.</a> Plant Physiol. 163, 216-231 (2013).</li><li>Wuest, S. E., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-assisted+microdissection+applied+to+floral+tissues.">Laser-assisted microdissection applied to floral tissues.</a> Methods Mol Biol. 1110, 329-344 (2014).</li><li>Wuest, S. E., Schmid, M. W., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+expression+profiling+of+rare+cell+types+as+exemplified+by+its+impact+on+our+understanding+of+female+gametophyte+development.">Cell-specific expression profiling of rare cell types as exemplified by its impact on our understanding of female gametophyte development.</a> Curr Opin Plant Biol. 16 (1), 41-49 (2013).</li><li>Wuest, S. E., 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=Arabidopsis+female+gametophyte+gene+expression+map+reveals+similarities+between+plant+and+animal+gametes.">Arabidopsis female gametophyte gene expression map reveals similarities between plant and animal gametes.</a> Curr Biol. 20, 1-7 (2010).</li><li>Cai, S., Lashbrook, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+of+plant+cells+from+tape-transferred+paraffin+sections+promotes+recovery+of+structurally+intact+RNA+for+global+gene+profiling.">Laser capture microdissection of plant cells from tape-transferred paraffin sections promotes recovery of structurally intact RNA for global gene profiling.</a> Plant J. 48 (4), 628-637 (2006).</li><li>Schmidt, A., Wuest, S. E., Vijverberg, K., Baroux, C., Kleen, D., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+analysis+of+the+Arabidopsis+megaspore+mother+cell+uncovers+the+importance+of+RNA+helicases+for+plant+germ+line+development.">Transcriptome analysis of the Arabidopsis megaspore mother cell uncovers the importance of RNA helicases for plant germ line development.</a> PLOS BIOL. 9, e1001155 (2011).</li><li>Schmid, M. W., Schmidt, A., Klostermeier, U. C., Barann, M., Rosenstiel, P., Grossniklaus, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+powerful+method+for+transcriptional+profiling+of+specific+cell+types+in+eukaryotes:+laser-assisted+microdissection+and+RNA+sequencing.">A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.</a> PLOS ONE. 7, e29685 (2012).</li><li>Mau, M., 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=Hybrid+apomicts+trapped+in+the+ecological+niches+of+their+sexual+ancestors.">Hybrid apomicts trapped in the ecological niches of their sexual ancestors.</a> Proc Natl Acad Sci.U S A. 112 (18), 2357-2365 (2015).</li><li>Aliyu, O. M., Seifert, M., Corral, J. M., Fuchs, J., Sharbel, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copy+number+variation+in+transcriptionally+active+regions+of+sexual+and+apomictic+Boechera.+demonstrates+independently+derived+apomictic+lineages.">Copy number variation in transcriptionally active regions of sexual and apomictic Boechera. demonstrates independently derived apomictic lineages.</a> Plant Cell. 25 (10), 3808-3823 (2013).</li><li>Corral, J. M., 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=A+conserved+apomixis-specific+polymorphism+is+correlated+with+exclusive+exonuclease+expression+in+premeiotic+ovules+of+apomictic+boechera+species.">A conserved apomixis-specific polymorphism is correlated with exclusive exonuclease expression in premeiotic ovules of apomictic boechera species.</a> Plant Physiol. 163 (4), 1660-1672 (2013).</li><li>Deeken, R., Ache, P., Kajahn, I., Klinkenberg, J., Bringmann, G., Hedrich, R. <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+Arabidopsis+thaliana.+phloem+RNAs+provides+a+search+criterion+for+phloem-based+transcripts+hidden+in+complex+datasets+of+microarray+experiments.">Identification of Arabidopsis thaliana. phloem RNAs provides a search criterion for phloem-based transcripts hidden in complex datasets of microarray experiments.</a> Plant J. 55, 746-775 (2008).</li><li>Schmid, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genome Scale Quantitative Biology in Arabidopsis thaliana. , 40-104 (2015).</li></ol>

The authors have nothing to disclose.

该协议适用于不同的细胞和组织类型 林与转录结合由微阵列或分析RNA测序是洞察基因活性的调节发育或生理过程7-11,13,14特定模式的有价值的工具。然而，该方法对于任何给定细胞类型的适宜性是极其依赖于结构问题。细胞必须清晰可见，并用于LAM干节明确识别。在Boechera divaricarpa，配子体的形态允许易于识别的卵细胞（ 图2，图3）。在结束时，一个特定细胞群体的分离的速度也取决于在该细胞类型可以在一个幻灯片中找到，作为交换幻灯片和扫描新的幻灯片的频率是耗时的步骤。给出依赖于细胞类型，至少200 - 250细胞节离子应每个样品被汇集，对一定的研究设计的方法的适宜性在很大程度上取决于为LAM步骤的时间要求。 此外，根据不同的组织的形态，它可能是有利的结构方面的部分的厚度减少至6微米。类似地，理想地≥8微米厚的部分通常会导致从相同量的组织切片的略微更高的品质更高的RNA数量和RNA。此外，所关注的细胞应具有至少一个直径为8 - 10微米的由LAM可行使隔离。 原则上，此处描述的方案可以进行调整，以来自远缘种不同的细胞和组织类型。然而，RNA的质量甚至不同类型的细胞相同物种7,10,13的之间变化。基于小和关键细胞类型此协议进行了调整。因此，现在可以应用到broade样品的R档而无需进一步的调整。协议的轻微的修改可能仍然是需要的，这取决于所研究的细胞类型和物种。建议先使用这两种替代固定剂（见1.1协议）设置为一个新的物种或细胞类型的形态和RNA质量的比较方法时。 原则上，该协议可以适于和用适于激光显微切割9个不同的仪器进行的。然而，需要注意的是，仪器在采样的技术变化都在激光功率和可应用的激光束的最小宽度，以及。特别是小细胞和组织的区域，激光产生一窄激光路径的隔离更适合。我们使用的仪器的激光束是相当精细（约1微米，宽峰），并允许小细胞清扫。收集CEL的采样技术上通过静电粘附粘帽表面LS为小细胞类型和单细胞部分的隔离是特别有利的。这最大限度地减少由静电效应样品损失的风险。 获得的RNA质量是进一步转录分析要点 一个成功的RNA测序文库制备中最关键点之一是RNA的质量。而扩增试剂盒的几个提供商优化他们的技术为甚至更低的完整性的RNA，由任一微阵列或RNA测序转录组分析后得到的数据的质量通常与输入RNA的质量增大。使用该建立的协议，得到为女性生殖组织的不同发育阶段好和高度可再现RNA的质量和在拟南芥和Boechera种系（ 例如， 图4）。而该方法是适用也为更远相关的物种（ 例如，西红柿，未发表），它需要考虑到小的优化或修饰可以取决于物种，组织类型是需要的，并且甚至在实验室环境，例如，在高湿度幻灯片准备和LAM期间可能影响RNA的完整性。 协议中的一个关键步骤是包含所述样本以幻灯片的剖蜡条纹的安装。这里，特别是接触到水是关键的一步。而由乙醇代替水不会在后隔离（未公布的）RNA的完整性的任何显著改善造成的，由甲醇置换水一样。然而，甲醇应仅在化学罩和非常谨慎处理。根据样品类型，作为替代使用甲醇，用水安装后的干燥时间可以减少到〜2小时（见3.3.3.1），产生同样好的RNA的质量。执行试验，以测试由安装无论是在水或甲醇的细胞类型和感兴趣的物种获得的RNA的质量，建议在一个新的项目的开始。 LAM是转录分析一个强大的技术 总之，我们已经成功地进行了优化，并施加LAM和通过微阵列和RNA测序，以在拟南芥中Boechera属性和无融合生殖种系谱系不同细胞的细胞类型特异性转录组分析的结合。，因而允许比较转录分析（也参见图5）7,11,13,14。相比于其它技术允许细胞类型特异性转录分析所描述的方法承担许多优点。重要的是，根据不同的细胞类型或在部分兴趣组织的识别性，它可以应用于稀有细胞类型都模型的和非模型物种，如成熟雌配子体在Boechera属的细胞，或者它甚至可以用于分离细胞的域， 例如 ，将细胞19的极性半部。类似的应用将是不可能与依赖于细胞或细胞核（FACS /风机）10的荧光激活分选等方法。 该方法的主要缺点是时间要求。而固定和嵌入并不需要延长动手时，可同时被用于大量的花，林完成，因此是非常耗时，并且用于小区类型特定的分析，通常为3天〜3周LAM（上在激光显微镜平均每天5小时）需要规划。在这方面，它是有帮助的有针对性的正确设计的研究中的特定细胞类型的分离的速度做一些预试验。另外，使用这种优化的协议，即使，试验，以测试该RNA的质量高度重新表彰。 总之，林是用于在单个细胞类型或甚至细胞子域的分辨率，这不能使用替代现有的方法来实现的转录分析的有力工具。在这方面，它承担巨大的潜力，以允许分析， 例如，发育过程中，具有非常高的时间和空间分辨率。这是由细胞的转录组在拟南芥和Boechera 7,11,13,14性与生殖无融合生殖的所有关键步骤的分析例证。

在组织水平进行转录的研究，高度专业化但罕见的细胞类型的转录由更丰富周围细胞通常掩蔽。对于这样的高度特化的细胞类型的例子是在植物中女性生殖谱系（种系）的细胞。女性生殖正在开发胚珠中指定的，种子的前身花1,2的雌蕊内。的大孢子母细胞（MMC）是女性生殖系的第一个单元。它经过减数分裂形成大孢子减少了四分。通常情况下，只有这些大孢子的一个生存和有丝分裂不分裂细胞分裂， 即在一个合胞体。这些有丝分裂之后是细胞化以形成成熟配子体，其典型地包括四个细胞类型:三级antipodals，二助细胞，卵，和中央细胞。鸡蛋和中央细胞是获得由两个精子细胞中斗受精雌配子BLE受精以产生显影种子1,2的胚和胚乳。在性模型系统拟南芥中，只有约50种子每花发展而大约50 - 80种子每花发展密切相关的属Boechera。因此，女性生殖系仅由少数高度特化的细胞类型的，使得它的极好的模型来研究发育过程，诸如细胞说明书和分化。 此外，见解执政植物繁殖的基因调控过程中可以应用的价值。在植物中，可能会出现通过种子（无融合生殖）两种性与无性繁殖。而有性生殖群体中产生遗传多样性，无融合生殖导致形成克隆的后代是遗传上相同母株。因此，无融合生殖对农业和种子生产应用中的巨大潜力，因为即使是复杂的孕产妇的基因型几代3,4,5维持不变。因为无融合生殖不天然存在于任何主要作物物种，无融合生殖的作物的工程是极大的兴趣3,4,5。然而，这个长期目标是难以实现的，因为无融合生殖的潜在的遗传和分子基础是不足够详细6理解。 要深入了解转录的基础上使用激光辅助显微切割（LAM）和下一代测序（NGS）治无融合生殖繁衍，细胞类型特异性转录谱是一个非常有效的方法7,8。 LAM已先行建立了动物和生物医学研究。在过去的几年中一直LAM也被应用到植物生物学6,9,10。在与其它的方法使单个细胞和组织类型的分析，林不需要的标记线6,9,10的产生。因此，它可以是应用谎称，恕不另行分子知识的任何细胞或组织类型。林的另一个优点是，它可以使用，只要适用于任何类型的细胞作为细胞可以在干燥部分基于位置和/或结构特征进行识别。林具有附加的优点，即使用固定的组织，以防止在加工过程中的转录轮廓的变化。 的兴趣， 例如，花组织的组织，在石蜡包埋之前固定在非交联固定剂。在石蜡包埋可以手动完成，以下建立的协议9,11。然而，对于脱水和渗透的使用自动化组织处理器的与蜡通常导致更高的再现性在RNA的质量和组织形态的保护方面。包埋在树脂组织的替代策略也已成功地由LAM 8用于细胞类型特异性的分析。然而，使用一个奥波的在蜡包埋组织ated处理器是非常有效的时间，因为许多样品可在一次需要动手的时间最少的处理。而RNA的质量通常不显著损失固定和嵌入过程中发生，用切片机薄片的制备，尤其，安装在用于林的frameslides仍然是RNA的质量的保存的关键步骤。此以前已注意到与使用的磁带传送系统的被描述为导致更好的RNA的质量在此步骤12。然而，这增加了制备该幻灯片的期间额外耗时的步骤，也需要特殊的设备。下面所描述的优化的协议可重复产生的RNA是足够的质量与基因芯片和新一代测序（NGS）接近7,11,13,14转录谱。此外，与所使用的激光显微切割显微镜，分离的细胞类型的高纯度是击溃inely生产7,11,13,14。 属Boechera是研究无融合生殖的关键步骤一个很好的模型系统。在Boechera，各种不同的性和无融合生殖种质的已经确定，并可以用于比较分析15,16,17。在细胞的细胞类型特异性转录的从阴种系免受性拟南芥和无融合生殖Boechera的比较，我们鉴定的基因和途径被差异表达，从而鉴定调节过程的新的方面理事无融合生殖7。此外，本研究证实林的细胞类型特异性的转录小和稀有细胞类型分析的适用性。我们已经使用该协议为不同的细胞类型中的各种植物物种的分析，但物种和组织特异性的修改的协议可能在某些情况下是需要的。

<table>
	<tbody>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>1,009,861,000</td>
			<td>absolute EMPROVE Ph Eur,BP,USP</td>
		</tr>
		<tr>
			<td>15 ml falcon centrifuge tubes</td>
			<td>VWR</td>
			<td>62406-200</td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Bioanalyzer</td>
			<td>Agilent</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Applichem</td>
			<td>A3686,2500</td>
			<td>100% Molecular biology grade</td>
		</tr>
		<tr>
			<td>Ambion Nuclease free water</td>
			<td>life technologies</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>ASP200 S</td>
			<td>Leica</td>
			<td>14048043624</td>
			<td>tissue processor&#160;</td>
		</tr>
		<tr>
			<td>black cardboard</td>
			<td></td>
			<td></td>
			<td>can be purchased in special paper shops</td>
		</tr>
		<tr>
			<td>DNA- and RNAse-free Frame Slides</td>
			<td>Micro Dissect GmbH</td>
			<td></td>
			<td>1,4 &#181;m PET-membrane; can also be purchased from Leica</td>
		</tr>
		<tr>
			<td>Dumond Forceps</td>
			<td>Actimed</td>
			<td>0208-5SPSF-PS</td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol lamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>exsiccator</td>
			<td>Sigma-Aldrich</td>
			<td>Z354074-1EA</td>
			<td>Nalgene Vaccuum Dessicator or similar equipment</td>
		</tr>
		<tr>
			<td>filter tips 10&#160; &#181;l&#160;</td>
			<td>Axon Lab AG</td>
			<td>AL60010</td>
			<td>can be replaced by similar tips</td>
		</tr>
		<tr>
			<td>filter tips 1000&#160; &#181;l&#160;</td>
			<td>Axon Lab AG</td>
			<td>AL60010</td>
			<td>can be replaced by similar tips</td>
		</tr>
		<tr>
			<td>filter tips 20&#160; &#181;l&#160;</td>
			<td>Axon Lab AG</td>
			<td>AL60020</td>
			<td>can be replaced by similar tips</td>
		</tr>
		<tr>
			<td>filter tips 200&#160; &#181;l&#160;</td>
			<td>Axon Lab AG</td>
			<td>AL60200</td>
			<td>can be replaced by similar tips</td>
		</tr>
		<tr>
			<td>forceps precision</td>
			<td>VWE</td>
			<td>232-1221</td>
			<td></td>
		</tr>
		<tr>
			<td>glass slide holder</td>
			<td>Huber &#38; Co.AG</td>
			<td>10.0540.01</td>
			<td>F&#228;rbek&#228;sten nach Hellendahl</td>
		</tr>
		<tr>
			<td>glass staining trough</td>
			<td>Huber &#38; Co.AG</td>
			<td>10.0570.01</td>
			<td>F&#228;rbekasten</td>
		</tr>
		<tr>
			<td>Heated Paraffin Embedding Module Leica</td>
			<td>Leica</td>
			<td>Leica EG 1150 H</td>
			<td>blocking station, similar devises are suitable</td>
		</tr>
		<tr>
			<td>Heating and Drying Table</td>
			<td>Medax</td>
			<td>15501</td>
			<td>other models and/or suppliers are suitable</td>
		</tr>
		<tr>
			<td>ice bucket</td>
			<td>VWR ice bucket with lid&#160;</td>
			<td>10146-184</td>
			<td>similar buckets equally suitable</td>
		</tr>
		<tr>
			<td>light table</td>
			<td>UVP An Analytical Jena Company</td>
			<td>TW-26&#160;</td>
			<td>white light transluminator</td>
		</tr>
		<tr>
			<td>microscope slide</td>
			<td>Thermo Scientific</td>
			<td>10143562CE</td>
			<td>cut edges</td>
		</tr>
		<tr>
			<td>microtome blade</td>
			<td>Thermo Scientific</td>
			<td>FEAHS35</td>
			<td>S35 microtome blade disposable</td>
		</tr>
		<tr>
			<td>MMI Cell Cut Plus Instrument</td>
			<td>MMI (Molecular Machines and Industries)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Non-stick, RNAse free Microfuge tubes, 2ml</td>
			<td>life technologies</td>
			<td>AM12475</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast X-TRA</td>
			<td>Roth</td>
			<td>X882.2</td>
			<td>for histology</td>
		</tr>
		<tr>
			<td>PicoPure RNA Isolation Kit</td>
			<td>life technologies</td>
			<td>KIT0204</td>
			<td>Arcturus PicoPure RNA Isolation Kit</td>
		</tr>
		<tr>
			<td>plastic balancing trays</td>
			<td>Semadeni AG</td>
			<td>2513</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic box</td>
			<td>Semadeni AG</td>
			<td>2971</td>
			<td>Plastikdose PS</td>
		</tr>
		<tr>
			<td>plastic lid for heating plate</td>
			<td></td>
			<td></td>
			<td>homemade</td>
		</tr>
		<tr>
			<td>preparation needle</td>
			<td>VWR</td>
			<td>631-7159</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA 6000 Pico Kit</td>
			<td>Agilent</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse free microfuge tubes</td>
			<td>life technologies</td>
			<td>AM12400</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse ZAP Decontamination Solution</td>
			<td>life technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi-automated Rotary Microtome&#160;</td>
			<td>Leica</td>
			<td>RM2245</td>
			<td>similar devises are equally suitable</td>
		</tr>
		<tr>
			<td>Tissue Loc&#160; Histo Screen Cassettes</td>
			<td>Thermo Scientific</td>
			<td>C-1000_AQ</td>
			<td>similar cassettes of other suppliers are suitable</td>
		</tr>
		<tr>
			<td>Tubes with adhesive lid, without diffusor 500 &#181;l&#160;</td>
			<td>MMI (Molecular Machines and Industries)</td>
			<td>50204</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylol (Isomere) ROTIPURAN</td>
			<td>VWR</td>
			<td>4436.2</td>
			<td>min. 99 %, p.a.,ACS, ISO SP</td>
		</tr>
		<tr>
			<td>process embedding cassettes</td>
			<td>Leica</td>
			<td>14039440000</td>
			<td>Leica Jet Cassette I without lid</td>
		</tr>
		<tr>
			<td>Universal Oven</td>
			<td>Memmert</td>
			<td>UF55</td>
			<td>other models and/or suppliers are suitable</td>
		</tr>
	</tbody>
</table>

laser-assisted microdissection (lam) as a tool for transcriptional profiling of individual cell types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

样品制备和LAM在连续的步骤完成 需要一些连续的步骤来制备的RNA用于通过林（ 图1）从选择的细胞类型的转录分析。此与花和直接固定在收获开始，以确保RNA群保持收获后保持不变。将组织包埋，切片，并固定在载玻片上。这允许细胞LAM和细胞类型特异性区段上的一个或多个盖（ 图1）汇集的分离。该材料可随后被冻结或RNA提取直接处理。除了LAM的优点是适用于细胞类型特异性的两个模型和非模型物种分析，该方法仍然是相当耗时。工作时间，有时更多的至少一周，需要用于从钛的步骤ssue收获的RNA提取（ 图1）。它需要被考虑，往往收获超过林的几天部分在一种生物样品和RNA提取汇集帐户。对于Boechera卵细胞，强烈推荐使用每个样品至少〜200段，具有高达100〜路段每天隔离。 感兴趣的细胞类型的分离取决于它们在干节能见度 细胞类型特异性的部分的隔离是协议的最耗时的步骤。到目前为止，还没有这步骤的自动化已经开发由于样品的复杂性。的使用软件工具感兴趣的细胞的识别是不可行的，因为干部分具有低对比度，并且由于该胚珠以不同角度瓦特定向的事实的样本之间的部分平面变化ithin所述组织（ 图2）。然而，成熟雌配子体与助细胞，卵细胞，中央细胞（反足细胞受精前退化）的独特的形态允许由研究人员卵细胞（ 图2B，C。;图3）的明确识别。事实上，在Boechera divaricarpa，卵细胞常常收缩制备期间一个位，因此从周围组织中分离，有利的卵细胞群的高纯度（ 图 2B，C。;图3）的隔离的特征。 林后帽的目视检查推荐 LAM后帽表面的目视检查允许样品， 例如 ，任何可能的污染的鉴定，通过灰尘。此外，它是有帮助的，以确保本身ctions粘在帽，如偶尔部分获得在手术过程中丢失。通常情况下，许多路段选择， 例如 ，卵细胞，可在一个盖几个小时LAM后看到。 既定的工作流程重复性产生良好的RNA质量 从雌性配子体细胞类型特异性林隔离，总RNA的量仅在低纳克范围。 RNA的这个限制量使得有必要使用从较大每个幻灯片为LAM后RNA的质量控制的近似的组织中分离出一个代表性的控制。这是通过细胞类型特异性RNA的从B的比较表明divaricarpa卵细胞相比，从同一载玻片分离较大的组织区域，指示相似的RNA质量控制（ 图4A，B）。使用这种方法，有良好的与RIN号＆＃高品质的RNA8805; 7可重复获得。取得的RNA的质量适合于细胞类型特异性RNA测序，导致在无融合生殖卵细胞表达236个基因的鉴定，但不是在无融合生殖中央细胞，助细胞，或无融合生殖初始小区，也不在细胞或拟南芥的成熟性配子体（ 图5），7。 <img alt="图1" src="/files/ftp_upload/53916/53916fig1.jpg" /> 图1:在协议的步骤的计划，表示每一步所需的最小时间固定的花朵或感兴趣的组织是一蹴而就。第二天，嵌入被启动，它运行过夜，导致嵌入材料在协议的第3天。在当天就可以产生从包埋标本蜡块开始3切片机切片。薄石蜡切片的色带安装在滑动并放置干燥过夜。上E要LAM几天将需要获得足够的材料为一种生物重复。 RNA分离和质量控制后，可进行对RNA测序文库制备准备转录组分析。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53916/53916fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53916/53916fig2.jpg" /> 图2:卵细胞在薄的部分清晰可辨，由于雌配子体的形态特征 （一）成熟的雌配子体（ 蓼型）的示意图（B - C）通过胚珠包藏在雌性配子体薄切片。 Boechera divaricarpa。卵细胞都清晰可见。由于雌配子体的形态往往不是所有类型的细胞（卵细胞:E，助细胞:■，中央细胞:CC）是在一个单节可见。比例尺= 50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53916/53916fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53916/53916fig3.jpg" /> 图3. Boechera divaricarpa。A 的卵细胞 和C B的成熟配子体科LAM divaricarpa在和，B和D前7微米，与激光设备（卵细胞:E，助细胞:S，中央细胞:CC）显微切割后。比例尺= 50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53916/53916fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53916/53916fig4.jpg" /> &lt;STRONG&gt;图4.激光解剖卵细胞和控制部分RNA质量。从卵细胞部分分析相比，从相同的幻灯片作为对照收获（B）更大的组织区域（A）RNA质量。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53916/53916fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53916/53916fig5.jpg" /> 图5.标识只有在无融合生殖卵细胞表达基因相比，无融合生殖和性成熟的配子或单元格的无融合生殖初始小区。在无融合生殖卵细胞表达的基因236，但既不在中央无融合生殖细胞，细胞助热图，B的无融合生殖或初始细胞gunnisoniana也不A的成熟性配子体的细胞拟南芥 7分层样本和基因的聚类是基于欧氏距离和层次凝聚的聚集。红色表示高表达和黑色低表达。颜色被每行缩放。 APO:无融合生殖<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53916/53916fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types at JoVE.com

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

激光辅助显微切割（LAM）作为个体细胞类型的转录谱的工具

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

laser-assisted-microdissection-lam-as-tool-for-transcriptional

Nanyang Technological University

Research

JoVE Journal

Biology

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

9.2K 次观看.  University of Zürich & Zürich-Basel Plant Science Center. Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera. 

视频: Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

激光捕获显微切割哺乳动物组织

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

科研

生物学

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

作为寄主植物挥发物的筛选工具electroantennographic生物活性

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

糖基谱研究植物细胞壁聚合物，用微阵列

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

使用高含量显微镜的细胞氧化还原分析

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

tchip-seq: 细胞专用表观体分析

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...