作者感谢S.哈克对正在进行的合作，推动就叶舌发展的讨论。这项工作是由美国国家科学基金会资助MCB 105205​​1和IOS-1848478的支持。

注意:在同一时间，组织被固定LM修复组织学分析的组织。检查形态特征，将引导后期LM染色切片。当比较突变体与野生型，执行原位杂交或免疫定位以限定的目的基因表达的结构域（在这种情况下，LG1）。 1.组织固定和处理<ol><li>种植玉米苗单位两周标准条件下，6岁。 </li><li>解剖横向截面（ 图4）茎尖。 <ol><li>消费税只是育苗土壤线以下。 </li><li>用刀片，从杆（削减1， 图4A）的底部除去薄片直到被一个或两个成熟叶片包围秆的椭圆是可见的（ 图4B）。 </li><li>进行另一个切割约10mm基部上述（切割2， 图4A）。这10毫米段将包含SAM和年轻的叶原基。 </li><li>转10mm的段，因此基本朝上。使平行于横向轴线两个切口，使得获得的组织2-3毫米厚的片（切口3和4中， 图4B）。丢弃外两部分，并保留中心切片进行固定并包埋。 注:菜帮可能会被裁剪并丢弃。 </li></ol></li><li>修复组织和流程嵌入。 <ol><li>确保在随后的步骤中使用的所有材料都是无RNA酶的。治疗与焦碳酸二乙酯（DEPC）溶液（每升溶液1 DEPC。偶尔晃动，并且高压釜孵育过夜）。烘烤玻璃器皿在200℃的烘箱或至少6小时更高和治疗塑料制品用RNase去污溶液。 </li><li>第1天:在冰上的玻璃小瓶浸入组织切片〜10毫升农民的修复（醋酸3:1乙醇）。所有样品都被解剖后，将VACUU米到除去气泡和固定剂的助剂渗透。真空下保持10分钟，然后释放真空慢。更换固定液，并在4℃下轻轻摇动孵育过夜。 </li><li>第2天:孵育以下一系列的解决方案，〜每次10毫升，每次1小时，所有轻轻摇动;在4℃下，在4℃下，在4℃下，在4℃下，在4℃下100％的乙醇100％的乙醇100％乙醇95％乙醇，1 85％乙醇:1乙醇:在室温下二甲苯，在室温下100％二甲苯中，在室温100％二甲苯。 注:二甲苯是通过接触和吸入有毒。在通风橱中工作，并使用合适的手套。 </li><li>石蜡组织包埋剂粒料添加到二甲苯的大约一半体积，并在室温轻轻摇动孵育过夜。 </li><li>第3天:所述小瓶转移到60℃的烘箱直至粒料熔化。倒出溶液，用新鲜融化的组织包埋剂代替。改变介质两次白天。 </li><li>第4天:改变组织在上午一度包埋剂。回到60℃烘箱中，直到下午。 </li></ol></li><li>铸造块<ol><li>将嵌入模具上嵌入组织站的热点板块。使用镊子组织样本转移到用切面朝下嵌入模具。顶部与熔化石蜡模具，并放置在嵌入环在模具的顶部。转移到冷板，直到石蜡凝固。存储在用硅胶密闭容器在4℃的石蜡块。 </li></ol></li></ol> 2.切片和幻灯片准备<ol><li>削减切片机25 10微米的部分。 </li><li>检查色带，并选择中间部分。位部分是包括在SAM，显示为通过叶原基包围细胞的圆顶。 </li><li>在幻灯片上安装的部分。 <ol><li>地方幻灯片适合于LM（无论是无RNA酶的或烤）上42℃下滑动温暖及申请的50％乙醇溶液几滴以覆盖滑动。 </li><li>浮在乙醇溶液部分，直到部分扩大。 注:乙醇溶液，而不是浮在水面的部分保持RNA的沉淀状态，减少RNA降解。 </li><li>倾斜滑动并取下吸过量的乙醇溶液用一次性移液管。使用不起毛的抹布吸走任何额外的乙醇溶液。 </li><li>在42℃下干燥载玻片数小时或过夜。商店在用硅胶气密容器滑动于4℃。 </li></ol></li><li> Deparaffinize上滑动，使用的当天。 <ol><li>准备包含三个玻璃罐子科普林; 100％二甲苯（二甲苯I）中，100％的二甲苯（二甲苯Ⅱ），和100％的乙醇（〜各50毫升溶液）。 </li><li>用清洁的镊子转移幻灯片，在二甲苯浸泡幻灯片我2分钟，二甲苯二为2分钟，1分钟100％的乙醇。 </li><li>排水下滑在室温下无绒抹布和空气干燥上课。 </li></ol></li></ol> 3.从Plastochron 7叶原基叶片，叶舌和护套样品显微切割<ol><li>安全LM显微镜阶段的幻灯片。使用五六个幻灯片每个重复，利用每张幻灯片五个部分。 注:组织汇集为一个复制在图5中示出。 </li><li>检查幻灯片和识别每张幻灯片上五个最中间的部分，采用了SAM顶点为中心参考点。 注意:此可在低倍率进行，通常是5X目标是足够的。 </li><li>使用10X或20X的目标，识别每个部分的plastochron 7叶原基的叶舌的位置。叶舌会从叶原基的近轴面突出凹凸可见。标记使用LM软件的绘图工具这一立场;选择铅笔图标，将光标移动到相应的POSITI上单击并拖动鼠标绘制。 注:10X 20X或目标是合适的，这和后续步骤。当使用侧部，每个叶原基的两侧将存在于每一个部分（ 图2A）。 </li><li>使用标尺工具和矩形绘图工具，测量100微米中心上每一节的叶舌高矩形（红色矩形， 图2A，2B）。这些都将是"叶舌"样本。 <ol><li>要使用标尺工具;选择标尺图标，将光标移动到物体的一端进行测量，单击并拖动来衡量的对象。尺子的长度将在屏幕上显示。 </li><li>要绘制矩形;选择矩形图标，将光标移动到一个点，这将是矩形的一个角落里，单击并拖动画出适当大小的矩形。或者，选择直线绘图工具，绘制四条直线。 </li><li>措施100微米的矩形定位为50μm以上和以下的"叶舌"矩形。 注意:这些将是"刀锋"和"鞘"的样品，分别为（绿色和蓝色的矩形， 如图2A，2B）。根据我们的组织学数据，一个100微米的矩形包含整个叶舌区域。被选为叶片和叶鞘的等效大小的部分，以保证采集组织类似数额为每个。为50微米间隔物使用，以确保没有叶舌区域组织无意中包含在叶片或鞘microdissections。 </li></ol></li><li> Microdissect测量矩形（ 图2D - 2F）7，8，9，收集叶舌，刀片和鞘样品在分开的管中。使用激光切割功能通过沿所选择的域的轮廓的组织部分进行切割。使用CATAPULT功能推动组织的矩形断滑动并进入管（ 图2D - 2F）的盖子。 </li></ol> 4.从显微切割7 Plastochron叶原基叶片，叶舌和鞘近轴表皮样的<ol><li>选择部分，并用尺子工具测量100微米高段集中在plastochron 7叶舌，在第3节（上图）描述。 <ol><li>通过使用绘图工具概述只选择每100微米高的"刀锋"和"鞘"段（绿色和蓝色选择， 图2C）的近轴表皮细胞。表皮是外细胞层;近轴侧是最靠近SAM中之一。 </li><li>对于"叶舌"的样品，选择如第3.3（红色选择， 图2C）中描述的新兴叶舌隆起只有细胞。 </li></ol></li><li> Microdissect选定区域，采集叶片，叶舌和乳木果日表皮分离的试管样品，如3.5节所述。 </li></ol> 5. LG1-R和野生型同胞显微切割Plastochron 6叶原基的<ol><li>分离生长突变体（LG1-R）和野生型植物的家庭。 </li><li>修复和处理茎尖为LM，如第1.2-1.4描述。修复，以保持他们单独野生型和单独的小瓶突变茎尖。从相同种植样品应固定和原位杂交处理以。 </li><li>确定LG1在野生型同胞转录， 通过原位杂交6，26，27执行LG1。测量从多个样本（ 图1E）在叶原基的基础LG1成绩单积累的位置。 </li><li> 原位杂交数据基础上，选择叶普里莫的一部分rdium即包括其中LG1被转录的区域。在这种情况下，从plastochron 6叶原基（紫色矩形， 图2A）的基400-900微米。 </li><li> Microdissect选择叶原基的部分，如3.5节所述，在单独的试管收集LG1-R和野生型样本。 </li></ol> 6.应用RNA提取液<ol><li>应用50微升RNA提取缓冲显微组织和RNA提取进行。用RNA提取，RNA扩增，文库构建，测序和生物信息学分析继续参考6描述。 </li></ol>

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The authors have nothing to disclose.

实验设计是在RNA-SEQ实验的关键因素。主要问题是精确域和发育阶段（多个）进行分析，和什么比较将进行。这是认为在比较而言是至关重要的，因为输出是通常被两个或多个条件之间DE基因的列表。如同所有的实验中，在一个时间，以改变只有一个变量是重要的。例如，比较不同的叶域时，叶片的相同年龄和发育阶段，在同样的条件下生长，应比较。 这些实验的目的是，以?...

玉米叶是研究形态发生过程发展领域的形成一个理想的模型，因为它具有在刀片和鞘是服从遗传解剖（ 图1A）之间有明显的边界。期间叶发育，较小的细胞的线性带的早期阶段，preligule带（PLB），细分叶原基成预刀片和预鞘域。刘海状叶舌和三角耳从PLB（ 图1A，C，D）制定。遗传筛选已经确定了破坏刀片鞘边界突变。例如，隐性liguleless1（LG1）突变删除叶舌和耳廓1，2，3，4（ 图1B）。 原位杂交透露，LG1成绩单积累的PLB和新兴叶舌，使其成为一个很好的标志物叶舌开发5，6（ 图1E）。 <img alt="图1" src="/files/ftp_upload/55004/55004fig1.jpg" /> 图1:野生型和 liguleless1-R 玉米叶片。 （A）显示叶舌和耳廓的结构成熟的野生型叶刃鞘边界区域。 （B）成熟liguleless1-R叶表现没有叶舌和耳廓结构的叶片，鞘边界区域。在A和B的叶子已经被削减了一半沿中脉。 （C）通过纵野生型叶原基部分。样本已被处理和染色用于组织学分析。发起叶舌是从叶（箭头）的平面中突出的凸起显而易见。 （D）纵向教派通过野生型叶原基离子。如在文本中所描述的样品已被处理为LM。箭头指示发起叶舌。 （E） 在LG1茎尖外侧纵截面的原位杂交。星号表示在P6叶原基的PLB LG1成绩单积累。箭头指示P6原基的基地。律师表示从原基小巴的基本测量。在A和B比例尺= 20毫米。在CE比例尺= 100微米。这个数字已经从6参考（植物生物学家著作权协会）修改。 <a href="http://ecsource.jove.com/files/ftp_upload/55004/55004fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 在这项研究中，LM RNA测序是采用叶片鞘边界相对于所述叶原基和IDE其他部分在识别基因的套件，差异表达（DE）<html> ntify基因是DE在LG1-R的突变体相对于野生型同胞。 LM RNA测序是在特定细胞或细胞结构域7定量转录物积累的方法。直线运动系统结合了激光器和用数码相机拍摄的显微镜。切片组织被固定在载玻片上，并通过显微镜观察。该软件LM通常包括绘图工具，允许用户在任何勾勒出选定的区域进行显微切割。激光切口沿着线，并且所选组织被弹射出滑动并进入悬挂在滑动上方的管中。 LM允许用户microdissect精确域，包括特定的细胞层和甚至单个细胞8,9。的RNA随后可以从显微组织中提取。随后，RNA测序组件利用下一代测序测序从提取的RNA 10产生cDNA文库，="外部参照"&gt; 11。 LM RNA测序的主要优点是能够量化精确定义的域成绩单的积累能力，来分析整个转录同时7的能力。该技术特别适合于探测早期发育事件，其中所述感兴趣区域是经常微观。先前的研究已利用的LM与微阵列技术相结合，研究在植物9，12，13的发育过程。 RNA测序具有在宽的动态范围，包括低表达基因定量转录物的优点，和现有序列信息不要求10,11。此外，LM RNA-SEQ有突出由于遗传冗余或到的杀伤力损失OF-可以在诱变筛选错过发育的重要基因的潜力功能突变体。 发育重要的基因，如窄护套1（NS1）和杯形cotyledon2（CUC2），通常具有的只是一个具体的表达模式或几个电池单元17，18，19，20。许多仅在早期发育阶段，而不是在成熟器官中表达。当整个器官或大结构域进行了分析，这些细胞特异性转录物稀释并可能无法在更常规的分析进行检测。通过允许精确限定域的分析，LM RNA测序使得这些组织特异性基因被识别和量化。 在这里描述的实验中的成功的关键因素是一个彻底的组织学分析是指导用于分析的合适的发育阶段和域的选择，以及精确measureme对于LM细胞组织域的NT。以确保等效域进行采样的所有重复，组织是从叶原基在相同发育阶段收集和显微结构域诸如新兴叶舌（ 图2），测定相对于形态的地标。已知的是某些基因在从尖端到叶的基部的梯度表示。通过测量精确域的变化，由于从沿叶近端-远端轴线的不同位置取样保持为最小（ 图3A）。由microdissecting大小相同的域，由于变异差分细胞特异性转录物的稀释也减少（ 图3B）。茎尖的外侧纵切片用于所有microdissections。这些是垂直于脉利润率轴线（ 图4）的部分。只使用部分包含了SAM确保能等效侧向地区叶原基进行了分析。 在处理和切片用于LM样品，叶舌向外生长的第一形态迹象是在近轴侧的凸点由于在近轴表皮（ 图1D， 图2）平周细胞分裂。它被确定新兴叶舌可以在plastochron 7阶段叶原基可靠地识别。我们感兴趣的整个叶舌区域表达的基因，包括新兴叶舌并将细胞立即远侧将形成耳廓。为了确保等效组织的选择进行了改造，所述叶舌凸点被用作形态学里程碑，被选定为LM（ 图2A，2B）中心的叶舌凸点100微米的矩形。从同一叶原基被预先选定的叶片和预鞘的等效尺寸矩形。 liguleless突变体的分析提出了不同的challeNGE; LG1-R突变体不形成舌，因此该形态特征不能被用于选择该区域的LM。相反，在野生型叶原基LG1转录积累的域被确定，并且被定义，将包含此结构域的区域。作为被用于最终分析这些初步分析在幼苗从同一种植进行的，因为以前的工作已经表明，PLB的位置取决于生长条件。 原位杂交表明LG1转录本P6的PLB的叶原基（ 图1E）累积。我们选择了一个域400-900微米从涵盖LG1表达式的域（紫色长方形， 图2A）和捕获野生型和LG1-R的植物，这些等效区域的叶原基的基础。为了比较转录时尽量减少遗传背景和生长条件的变化在LG1-R和野生型植物，分离的突变体和野生型同胞家属牛逼积累量使用。

<table><tbody><tr><td>Diethyl pyrocarbonate</td><td>Sigma-Aldrich</td><td>159220</td><td>Used for RNase treatment of solutions</td></tr><tr><td>Razor blades&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>72000</td><td></td></tr><tr><td>RNase Zap</td><td>Sigma-Aldrich</td><td>R2020-250ML</td><td>RNase decontamination solution</td></tr><tr><td>Ethanol absolute 200 proof</td><td>Fisher Scientific</td><td>BP28184</td><td></td></tr><tr><td>Acetic acid, glacial</td><td>Sigma-Aldrich</td><td>A6283</td><td></td></tr><tr><td>Glass vials - 22 ml</td><td>VWR</td><td>470206-384</td><td></td></tr><tr><td>Xylenes, histological grade</td><td>Sigma-Aldrich</td><td>534056-4L</td><td></td></tr><tr><td>Paraplast plus</td><td>Sigma-Aldrich</td><td>P3683-1KG</td><td></td></tr><tr><td>Disposable base molds, 15 mm x 15 mm x 5 mm</td><td>VWR</td><td>15154-072</td><td></td></tr><tr><td>Embedding rings</td><td>VWR</td><td>15154-303</td><td></td></tr><tr><td>Silica gel packets</td><td>Electron Microscopy Sciences</td><td>71206-01</td><td>Desiccant for storage of paraffin blocks</td></tr><tr><td>Oven</td><td>Fisher Scientific</td><td>15-103-0503</td><td>Oven must maintain temperature of 60 &amp;deg;C</td></tr><tr><td>Paraffin embedding station</td><td>Leica&amp;nbsp;</td><td>EG1160</td><td></td></tr><tr><td>Microtome</td><td>Leica&amp;nbsp;</td><td>RM2235</td><td></td></tr><tr><td>Slide warmer</td><td>Electron Microscopy Sciences</td><td>71317-10</td><td></td></tr><tr><td>Coplin jars</td><td>Electron Microscopy Sciences</td><td>70316-02</td><td></td></tr><tr><td>Laser microdissector</td><td>Zeiss</td><td></td><td></td></tr><tr><td>KIMWIPES&amp;trade; Delicate Task Wipers</td><td>Kimberly-Clark Professional</td><td>34120</td><td>Lint-free wipes for wicking excess solutions from microscope slides</td></tr><tr><td>Membrane Slide 1.0 PEN</td><td>Zeiss</td><td>415190-9041-000</td><td>Slides for laser microdissection</td></tr><tr><td>Adhesive Cap 200 opaque</td><td>Zeiss</td><td>415190-9181-000</td><td>Tubes for laser microdissection</td></tr><tr><td>PicoPure RNA Isolation Kit</td><td>ThermoFisher Scientific</td><td>KIT0204</td><td></td></tr></tbody></table>

experimental design for laser microdissection rna-seq: lessons from an analysis of maize leaf development

在发展重要作用的基因往往有空间和/或时间限制的表达模式。通常这些基因转录物不被检测或作为整个植物器官的转录分析差异表达（DE）不识别。激光显微切割RNA测序（RNA LM-SEQ）是一个强大的工具，以确定被取消的具体发展领域的基因。然而，蜂窝结构域的选择microdissect和比较，以及microdissections的精度是该实验的成功至关重要。在这里，两个例子说明了转录实验设计考虑;个LM的RNA-SEQ分析，以确定沿着玉米叶近端-远端轴DE基因，和第二个实验，以确定是-R liguleless1（LG1-R） 的 DE中的基因 突变体与野生型。 ，对这些实验的成功作出贡献的关键因素进行了详细的组织学和SI该区域的涂杂交分析待分析，在相当于发育阶段叶原基的选择，使用形态的地标选择区域用于显微切割，并精确测量畴的显微解剖。本文提供了通过LM RNA测序发育域分析详细的协议。此处呈现的数据说明了如何选择用于显微切割的区域会影响获得的结果。

使用图2所概述的LM方案中，约1,000,000-1,500,000微米2组织中收集的每个中的所有单元层LM复制（ 图5），200,000微米2每复制为近轴表皮LM。大约组织2,500,000微米2收集对每个在LG1-R和野生型叶原基的LM复制。两轮线性RNA扩增得到微克量的RNA的每个重复。从任何给定的显微解剖获得的RNA的量将依赖于细胞大小和转录活性以及所...

Watch this Scientific Journal Video about Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development at JoVE.com

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

激光显微切割RNA测序实验设计:从玉米叶片发展的经验分析

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

experimental-design-for-laser-microdissection-rna-seq-lessons-from-an

Research

JoVE Journal

Biochemistry

9.4K 次观看.  Cornell University. Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena. 

视频: Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

从外膜囊泡内蛋白质导演包装<em&gt;大肠杆菌</em&gt;:设计，生产和纯化

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

科研

生物化学

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

木质素下调<em&gt;玉米</em&gt;通过dsRNAi和Klason木质素分析

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

生物工程

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

激光显微切割的从细胞的一个子集基因表达分析中的应用果蝇永光盘

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

生物学

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.

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

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.

激光辅助显微切割（LAM）作为个体细胞类型的转录谱的工具

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

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of these analyses is of crucial importance, because growth is driven by cell division and cell elongation. Kinematic analysis represents a methodology to quantify these two processes. Moreover, this technique is easy to use in non-specialized laboratories. Here, we present a protocol for performing a kinematic analysis in monocotyledonous maize (Zea mays) leaves. Two aspects are presented: (1) the quantification of cell division and expansion parameters, and (2) the determination of the location of the developmental zones. This could serve as a basis for sampling design and/or could be useful for data interpretation of biochemical and molecular measurements with high spatial resolution in the leaf growth zone. The growth zone of maize leaves is harvested during steady-state growth. Individual leaves are used for meristem length determination using a DAPI stain and cell-length profiles using DIC microscopy. The protocol is suited for emerged monocotyledonous leaves harvested during steady-state growth, with growth zones spanning at least several centimeters. To improve the understanding of plant growth regulation, data on growth and molecular studies must be combined. Therefore, an important advantage of kinematic analysis is the possibility to correlate changes at the molecular level to well-defined stages of cellular development. Furthermore, it allows for a more focused sampling of specified developmental stages, which is useful in case of limited budget or time.

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Quantifying cell division and expansion is of crucial importance to the understanding of whole-plant growth. Here, we present a protocol to calculate cellular parameters determining maize leaf growth rates and highlight the use of these data for investigating molecular growth regulatory mechanisms by directing developmental stage-specific sampling strategies.

细胞分裂和扩张的运动学分析:量化增长和采样产业开发区的细胞学基础<em&gt;玉米</em&gt;叶

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

基于 X 射线 Microcomputed 层析成像的玉米血管束微米尺度分型技术研究

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

激光捕获微分块RNA-测序空间和时间组织特异性基因表达分析

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Laser Microdissection for Species-Agnostic Single-Tissue Applications

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic &#34;toolkits,&#34; such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis.
LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species.
Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.

A protocol is described that uses laser microdissection to isolate individual nematode tissues for RNA-sequencing. The protocol does not require species-specific genetic toolkits, allowing gene expression profiles to be compared between different species at the level of single-tissue samples.

激光显微切割，用于与物种无关的单组织应用

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific ...

改进的玉米叶片原基成像方法

Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging

In maize (Zea mays) and other grasses (Poaceae), the leaf primordia are deeply ensheathed and rolled within the leaf whorl, making it difficult to study early leaf development. Here, we describe methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging. The first method uses a wire stripper to remove the upper portions of older leaves, exposing the tip of the leaf primordium and allowing its measurement for more accurate transverse section sampling. The second method uses clear, double-sided nano tape to unroll and mount whole-leaf primordia for imaging. We show the utility of the two methods in visualizing and analyzing fluorescent protein reporters in maize. These methods provide a solution to the challenges presented by the distinctive morphology of maize leaf primordia and will be useful for visualizing and quantifying leaf anatomical and developmental traits in maize and other grass species.

作者聚焦：用于荧光和共聚焦成像的玉米叶片原基横向切片和展开整片的改进方法  

Maize leaf primordia are deeply ensheathed and rolled, making them difficult to study. Here, we present methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging.

用于荧光和共聚焦成像的玉米叶原基横向切片和展开整片的制备方法的改进方法

In maize (Zea mays) and other grasses (Poaceae), the leaf primordia are deeply ensheathed and rolled within the leaf whorl, making it difficult to study ...

激光显微切割RNA测序实验设计:从玉米叶片发展的经验分析

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木质素下调

激光显微切割的从细胞的一个子集基因表达分析中的应用果蝇永光盘

激光辅助显微切割（LAM）作为个体细胞类型的转录谱的工具

细胞分裂和扩张的运动学分析:量化增长和采样产业开发区的细胞学基础

基于 X 射线 Microcomputed 层析成像的玉米血管束微米尺度分型技术研究

激光捕获微分块RNA-测序空间和时间组织特异性基因表达分析

激光显微切割，用于与物种无关的单组织应用

用于荧光和共聚焦成像的玉米叶原基横向切片和展开整片的制备方法的改进方法

用于荧光和共聚焦成像的玉米叶原基横向切片和展开整片的制备方法的改进方法