Name: laser-capture microdissection of human prostatic epithelium for rna analysis
Uploaded: 2015-11-26T15:00:00.000+00:00
Duration: 7 min 42 s
Description: Watch this Scientific Journal Video about Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis at JoVE.com

We thank Dr. Vicky Macias, Angeline Giangreco and Avani Vaishnav for assistance with optimizing this methodology over the years and Yang Zhang and Dr. Jian Ma at the University of Illinois at Urbana-Champaign for the RNA seq analysis. This work was supported by NIH/NCI R01 CA166588-01 (Nonn), American Cancer Society Research Scholar 124264-RSG-13-012-01-CNE (Nonn), NIH/NCI R03 CA172827-01 (Nonn), DOD-CDMPR PRCP Health Disparities Idea Award PC121923 (Nonn) and a Prevent Cancer Foundation grant (Zhou).

通过机构审查委员会批准的协议和/或豁免在伊利诺伊大学芝加哥分校获得了用于这些实验的所有人体组织。 1.第新鲜冰冻前列腺到PEN-幻灯片，到带电玻片<ol><li>前一天或切片样前几个小时，清洁工具（即，刷，镊子，coplins，叶片，PEN框载玻片（如果不是已经无RNase），一个ETOH安全标记和铅笔）和一个内室温的低温恒温器，使用喷雾瓶和实验室湿巾RNase的去污溶液。 <ol><li>允许RNase的去污溶液坐5分钟擦去前，用depcH 2 0冲洗，以除去遗留RNase的去污溶液中，用70％乙醇的最终漂洗（制备有depcH 2 0）。 注意:重​​要的是要保持无RNase的工作区。所有coplins，工具，冻结的安装中，叶片和使用的幻灯片，应仅用于核糖核酸酶免费工作与从未使用在标准的非无RNA酶的环境。所有仪器必须与每个实验之前核糖核酸酶去污溶液进行处理。 </li></ol></li><li>低温恒温器冷却至-24℃。 </li><li>将下面的清洁无RNA酶的仪器内的低温恒温器，以至少30分钟冷却切片之前:滑动科普林填充了100％的乙醇，工具，小型冷冻组织盒和安装卡盘。 </li><li>检索cryostorage和地方冷冻前列腺组织成一个干冰填充泡沫桶运输。放置入组织冷冻切片机平衡到低温恒温器温度为至少30分钟。 注意:人类前列腺组织的激光捕获显微解剖可以是新鲜冷冻或福尔马林固定石蜡包埋（FFPE）。时间紧迫和幻灯片准备是组织源之间略有不同。在这里，我们描述的步骤进行冰冻切片。 </li><li>有一个组织在工作时间和喷射冰冻安装介质插入戈巴托使用冷冻镊子盒的米，并迅速装入冷冻的前列腺组织进入冰冻封固剂。 </li><li>放置与组织盒到冷却器中的低温恒温器5分钟以设定冷冻封固剂。 </li><li>喷射少量冰冻安装介质上的吸盘和高达小心地按上的组织下侧到冰冻的安装介质。让在低温恒温器设定为5分钟，以允许冷冻封固剂完全固化。 </li><li>将卡盘插入支架，并拧紧。 </li><li>锁定手柄锁定位置，并把新刀片到低温恒温器。为下游分子端点包括扩增步骤（即，定量PCR），建议使用一个新的刀片针对每个病人，以防止污染（或移 ​​动刀片转移到为下一个样品使用叶片的面积）。 </li><li>解锁手柄，通过用手或用脚踏小心推动组织到连和暴露组织用50μ米的部分，直到所需的组织暴露为止。 </li><li>调节低温恒温器至5μm，切一个或两个部分，并将其放置到充电载玻片用于苏木精和伊红（H＆E）染色（参见步骤2）。 </li><li>立即滑动放入冷100％乙醇2分钟。 </li><li>从乙醇中除去带电的载玻片上并让它干燥在RT前进到H＆E染色（步骤2）之前。 </li><li>将RT PEN帧幻灯片的帧支持。 </li><li>低温恒温器调整到10微米和地点节到RT PEN帧幻灯片。一到四个部分可以放置到根据组织规模每张幻灯片。 </li><li>立即沉浸滑动进入冷100％乙醇2分钟。 </li><li>从乙醇中取出笔帧幻灯片，并在幻灯片中的幻灯片存储在干燥器中在-80℃冷冻，直到准备LCM。三天的最大存储时间为最佳RNA回收。 </li><li>继续执行步骤2只收取玻璃滑即继续执行步骤3笔帧幻灯片。 </li></ol> 2. HE染色-Y（H＆E）染色的组织学标记机注:本如果在充电载玻片上组织不是LCM笔帧幻灯片。 <ol><li>通过梯度乙醇水合物浸渍幻灯片上带电载玻片的部分如下:在70％乙醇浸滑动在100％乙醇两次持续3分钟，在95％乙醇两次持续3分钟，一次3分钟和两个在蒸馏的 H 2 O时间3分钟。 <ol><li>制备酸醇溶液99毫升70％乙醇+ 1 ml的1％HCl和0.1％碳酸氢钠溶液:0.1克碳酸氢钠在100毫升蒸馏水H 2 O的</li></ol></li><li>浸在苏木吉尔II（0.4％），滑动5分钟。 </li><li>在稳定运行阶H 2 O 3分钟洗掉多余的污点。 </li><li>浸滑酸醇10倍</li><li>在自来水H 2 O的3分钟清洗的幻灯片。 </li><li>浸滑在0.1％的碳酸氢钠溶液30-60秒转动紫色颜色为蓝色。 </li><li>在自来水H 2 O的3分钟清洗的幻灯片。 </li><li>漂洗在95％乙醇的滑动用10骤降。 </li><li>浸滑的曙红-Y 2-3次，持续15秒的总。 </li><li>通过梯度乙醇脱水幻灯片如下:沾下滑95％乙醇的两倍，在100％的乙醇两次，两次二甲苯（确保它表示彻底脱水的组织出现明显的）。 </li><li>安装有非水永久硬封固剂和盖玻片的幻灯片。 </li><li>让滑动干燥30分钟。 </li><li>扫描幻灯片与任何整个幻灯片扫描仪和打印大型，高分辨率图像到8"×11"的纸张。 </li><li>感兴趣的领域纲要用记号笔（通常由一个委员会认证的病理学家完成）。此标记是LCM的程序指南。 </li></ol>笔帧幻灯片3.甲苯胺蓝染色注:钍在完成当天的LCM。使用depcH 2 O水中所有的解决方案。完成在无RNase区域的所有步骤。所有coplins必须用RNase-去污溶液进行清洗。 <ol><li> LCM的天除去从-80℃冷冻室和水合物的PEN-帧滑动通过缓倾滑入90％乙醇2分钟，然后75％乙醇2分钟。 <ol><li>制备0.5％甲苯胺蓝溶于在分子生物学级水，然后过滤灭菌，通过0.2微米的过滤器，并储存在室温。此溶液可以被重新使用长达6个月。注:强烈建议准备的溶液，作为过滤可以采取几个小时之前。 </li></ol></li><li>由缓倾滑入0.5％甲苯胺蓝5-30秒的染色前列腺组织。脱色通过洗涤所述组织滑动的2倍depcH 2 O，持续15秒，随后通过75％乙醇30秒至3分钟。注意:染色和脱色时间可以调整，以保证良好的染色。前列腺往往超过污点。 </li><l我&gt;传送PEN帧幻灯片RNA酶，无干货集装箱冰上。 </li></ol> 4.激光捕获显微解剖（LCM） <ol><li>紧接的LCM，在幻灯片在42℃温暖15分钟干滑动。干只将被处理，并在冰上其余存储直到准备为他们的幻灯片。 </li><li>打开LCM和计算机的顺序，显微镜功率，激光键，激光开关，然后计算机。启动激光显微切割软件。 </li><li>使用该软件，以控制显微镜加载幻灯片和收集管。点击"卸载样品架"，加载使用部分滑动到显微镜幻灯片持有人与组织朝下并插入在显微镜下的相应插槽幻灯片固定器，然后单击"继续"。 <ol><li>点击"卸载收集管"，标签和负载0.5 mL管到管座和在仪表相应的插槽中插入。一旦瓶盖固定，加入35微升裂解液的收集瓶盖，尽量避免气泡。然后单击"确定"。 注意:如果蒸发是一个问题，稀释缓冲如下:将25μl的裂解缓冲液与10微升depcH2O。 </li></ol></li><li>在软件中，选择幻灯片已经被放置在载片支架的位置。选择收集罩收集LCM标本。 </li><li>可视化和注重通过电脑幻灯片在10X。使用该软件通过选择"激光"，然后"校准"来校准激光。调整激光的功率，光圈和速度，选择"激光"，然后"控制"，以允许所述激光束切割完全和有效地选择的区域。重复调整，直到激光切割完全穿过组织。 </li><li>使用8"×11"病理学家加价为指导，用"画形"的工具，外接上皮的所需区域lium在视图中，并避免收集任何基质。 注:多个区域可以在一个视图中选择，但是不要点击"切形"工具移动到另一种观点认为不先切割。 <ol><li>如果一个地区没有得到完全由激光切割使用"移动和切"工具手动过目一下。继续移动物镜，以新的观点，并重复激光切割，直到滑动被完全切开或已采集的组织的所需量（通常为100良性腺体收率150-500纳克的RNA）。 （参见例如在图1A）。 </li></ol></li><li>小心地从收集架管和关闭帽，被铭记在帽裂解液和组织。短暂离心30秒，收集的液体和组织在管的底部。孵育样品在42℃进行30分钟，并储存于-80℃，直到准备用于RNA分离。 </li></ol> 5.总RNA分离与费尔之三为基础的工具包和质量分析<ol><li>解冻管在冰上，使溶液的体积为100微升。 </li><li>通过施加30微升ofLysis解滤波器的中心预润湿的RNA分离试剂盒的微过滤器，并允许它浸泡whileperforming下两步（至少5分钟）。 </li><li>加入3微升LCM添加剂溶液（试剂盒中提供），以裂解物并通过涡旋混合5秒。离心30秒在试管底部收集流体。 </li><li>离心预湿的过滤器〜30秒，最高时速去除液体。 </li><li>添加1.25体积的100％乙醇（在这种情况下，129微升）以裂解物混合物中，轻轻涡旋或吸管上下。 **此方法将恢复大和小RNA种类。 </li><li>加载样品到预润湿的微过滤器，和离心机在10,000rpm×g离心1分钟，以结合核糖核酸到过滤器。然后洗微过滤器180微升"洗涤液1，R21;离心在10,000rpm×g离心1分钟。 注:从这点上的所有离心应该13,000 XG，或最高速度来完成。 </li><li>洗涤用180微升"洗涤溶液2和3"，分别过滤两次的指示该试剂盒的协议。离心30秒，然后丢弃通过流，和自旋过滤器，持续1分钟，以除去残留的流体和以干燥过滤器。 </li><li>转移过滤到一个新的收集管。 </li><li>暖洗脱溶液至-95℃（由试剂盒提供）。 </li><li>申请10微升预热洗脱溶液在过滤器的中心，盖上并存储5分钟，室温。然后离心1分钟以洗脱RNA，并重复此步骤一次，得到〜20微升的RNA。 </li><li>进行DNA酶I处理15分钟，在37℃。 </li><li>量化的RNA通过分光光度计260nm处的吸光度。光密度在260毫微米和280毫微米波长的比率被用来评估的纯度的核糖核酸（ 见表 1）15。 </li></ol> 6.基因表达分析注意:长RNA种类（像的mRNA和lncRNAs）的基因表达被示出在步骤6.1和短RNA物质（如微RNA）示于步骤6.2。不同套件可用于RT-qPCR的和由试剂盒所需RNA的量而变化（如少至10毫微克）。 RNA测序分析在6.3节。 <ol><li>逆转录（RT）的RNA成cDNA混合5微升20毫微克/μLRNA样品与5微升RT主混合物（RT缓冲液，的dNTP的，随机引物，逆转录酶，RNA酶抑制剂）。孵育25分钟，反应在25℃，然后120分钟，在37℃，5分钟，在85℃。 </li><li>稀释RT反应1:10无RNA酶的水，并使用每10微升的qPCR反应2.5微升用引物或引物/探针组的目的基因。 注:为方便部分降解RNA分析最好是使用底漆FOř短扩增子（小于100bp）16（表2B） <ol><li>对于微小RNA表达分析运行混合5微升2-10纳克/微升RNA样品与5微升RT主结构的RT反应。 注意:每个市售基于PCR微小RNA检测技术需要使用的专有和特异性RT引物。 </li></ol></li><li>可替代地或除了RT-PCR，使用下一代测序（RNA测序或小核糖核酸-SEQ）来量化不同物种的RNA。通常情况下，使用500ng的RNA用于此过程（见表3）和实施该方法由生产和检测仪所指示的。 </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+six+RNA+extraction+methods+for+the+detection+of+classical+swine+fever+virus+by+real-time+and+conventional+reverse+transcription-PCR.">Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription-PCR.</a> Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc. 17, 574-578 (2005).</li><li>Kong, H., 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=Quantitative+assessment+of+short+amplicons+in+FFPE-derived+long-chain+RNA.">Quantitative assessment of short amplicons in FFPE-derived long-chain RNA.</a> Scientific reports. 4, 7246 (2014).</li></ol>

The authors have nothing to disclose.

基因表达的来自人的标本分析可以是具有挑战性的，不仅对质量或组织的可用量，同时也为存在于一个给定的组织标本的各种组织学实体。这是在其中良性组织基本上是间质组织和癌症领域是不含基质的前列腺特别具有挑战性。 LCM促进前列腺间质和上皮的RNA物理分离为两种不同的细胞类型（图1A）的更精确的签名。在比较macrodissection或全部组织处理，LCM可以分离细胞区室中良性组织?...

前列腺是一种异质性组织安排在腺体腺泡由肌纤维间质主要由平滑肌组成1包围分泌上皮细胞组成。上皮隔室由五个不同但有组织的细胞类型:基底细胞，分泌细胞，神经内分泌细胞，短暂扩充细胞和干细胞2。在前列腺癌（PCa），它产生于腔上皮细胞，腺癌的生长会导致基质室 3的一个明显的渐进性下降。由于这些原因，组织标本将具有在基质和上皮细胞类型的基于前列腺癌的程度的比例有明显的差异。这些差异会导致从整个组织，不考虑所期望的细胞类型的显微解剖获得的基因表达数据的偏颇的假设。因此，为了消除这种偏差，必须事先对RNA提取和分离的细胞类型基因表达分析。 Macrodissection或显微切割可用于物理地从周围基质4 -6分离充分表征的上皮区域。 Macrodissection典型的做法是与在解剖显微镜下用刀片和可以很好地用于分离大前列腺癌从基质结节，但是不能够从周围的基质中除去良性上皮的（参见实施例良性前列腺组织学于图1）。显微切割用激光（LCM）比macrodissection显著劳动密集型的，但可以非常精确地解剖良性上皮4。 从我们的实验室最近的出版物已经表明，核糖核酸可以成功地通过从任一福尔马林固定石蜡包埋（FFPE）的活检或冷冻的组织4,7-9 LCM萃取。在LCM-RNA提取的主要挑战是:1）精确解剖组织的所需区域，和2）以保持ř液晶显示模块和分离过程4,10中NA的完整性。从纯的细胞群中分离RNA可通过多种方法，包括反转录定量PCR（RT-qPCR的）7,8，微阵列11，和深-sequencing 12-14中可用于基因表达分析。 该协议的目的是从隔离LCM前列腺上皮的总RNA从冷冻的组织为下游基因表达分析。

<table><tbody><tr><td>RNase-AWAY&amp;nbsp;</td><td>MBP</td><td>7005-11</td><td></td></tr><tr><td>PEN-membrane 4.0 mm slides&amp;nbsp;</td><td>Leica</td><td>11600289</td><td></td></tr><tr><td>Glass slides, Superfrost Plus</td><td>FisherBrand</td><td>12-550-15</td><td></td></tr><tr><td>Ethanol 200 proof</td><td>Decon labs.</td><td>2701</td><td></td></tr><tr><td>DEPC (diethyl pyrocarbonate)</td><td>Sigma</td><td>D-5758</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM3050</td><td></td></tr><tr><td>Coplins (Staining jar)</td><td>IHCWORLD</td><td>M900-12</td><td></td></tr><tr><td>Coplins (Staining rack)</td><td>IHCWORLD</td><td>M905-12</td><td></td></tr><tr><td>Aperio ScanScope</td><td>Aperio(Leica)</td><td>ScanScope&amp;reg; CS</td><td></td></tr><tr><td>Toluidine Blue</td><td>Fluka</td><td>89640-5G</td><td></td></tr><tr><td>Laser Microdissection System</td><td>Leica</td><td>LMD7000</td><td></td></tr><tr><td>0.5 ml Thin-walled Tubes for LCM</td><td>Thermo Scientific</td><td>AB-0350</td><td></td></tr><tr><td>RNAqueous&amp;reg;-Micro Total RNA Isolation Kit</td><td>Ambion</td><td>AM1931</td><td>Thermo Fisher Scientific Brand</td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td>ND-1000</td><td></td></tr><tr><td>Qubit 2.0 Fluorometer</td><td>Life Technologies</td><td>Q32866</td><td>Thermo Fisher Scientific Brand</td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>Applied Biosystems</td><td>4368814</td><td>Thermo Fisher Scientific Brand</td></tr><tr><td>Universal cDNA Synthesis Kit II, 8-64 rxns</td><td>Exiqon</td><td>203301</td><td></td></tr><tr><td>TaqMan microRNA RT kit</td><td>Applied Biosystems</td><td>4366597</td><td>Thermo Fisher Scientific Brand</td></tr><tr><td>Hematoxylin stain&amp;nbsp;</td><td>Ricca Chemical Company</td><td>3536-16</td><td></td></tr><tr><td>Eosin-Y</td><td>Richard Allan Scientific&amp;nbsp;</td><td>7111</td><td></td></tr></tbody></table>

laser-capture microdissection of human prostatic epithelium for rna analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

在先前的研究中，我们展示了采用LCM来收集上皮细胞和间质组织由基因和微RNA的RT-qPCR的冷冻和FFPE前列腺组织来自同一患者4比较表达谱。 LCM非常耗时，尤其是当大量的RNA被收集新一代测序分析。因此，关键的是要保留的工作空间和工具无RNA酶。建议检查收集（更详细地在下面的段落中讨论）的RNA的质量和细胞特异性的控制。 图1显示之前和之后激光俘获良性上皮在显微镜下PEN...

Watch this Scientific Journal Video about Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis at JoVE.com

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

人类前列腺上皮的激光捕获显微切割的RNA分析

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

laser-capture-microdissection-human-prostatic-epithelium-for-rna

Research

JoVE Journal

Medicine

13.4K 次观看.  University of Illinois at Chicago. The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

视频: Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

科研

医学

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results in cancer regression. However, in a significant number of cases, the disease progresses to advanced, castration-resistant prostate cancer (CRPC), which has limited therapeutic options and is often aggressive. Distant metastasis is mostly observed at this stage of the aggressive disease. CRPC is treated by a second generation of AR pathway inhibitors that improve survival initially, followed by the emergence of therapy resistance. Neuroendocrine prostate cancer (NEPC) is a rare variant of prostate cancer (PCa) that often develops as a result of therapy resistance via a transdifferentiation process known as neuroendocrine differentiation (NED), wherein PCa cells undergo a lineage switch from adenocarcinomas and show increased expression of neuroendocrine (NE) lineage markers. In addition to the genomic alterations that drive the progression and transdifferentiation to NEPC, epigenetic factors and microenvironmental cues are considered essential players in driving disease progression. This manuscript provides a detailed protocol to identify the epigenetic drivers (i.e., small non-coding RNAs) that are associated with advanced PCa. Using purified microRNAs from formalin-fixed paraffin-embedded (FFPE) metastatic tissues and corresponding serum-derived extracellular vesicles (EVs), the protocol describes how to prepare libraries with appropriate quality control for sequencing microRNAs from these sample sources. Isolating RNA from both FFPE and EVs is often challenging because most of it is either degraded or is limited in quantity. This protocol will elaborate on different methods to optimize the RNA inputs and cDNA libraries to yield most specific reads and high-quality data upon sequencing.

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

从卡西甲抗性前列腺癌患者的甲酸固定组织和血清衍生的外源体对小型非编码RNA进行测序

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results ...

癌症研究

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

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

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

应用生物标志物发现在前列腺癌：个别赫尔维位点表达微阵列鉴定

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

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

发育生物学

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

在前列腺癌的临床组织中miRNA的表达分析

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

神经科学

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

从细胞亚群特异性RNA提取使用激光捕获显微切割加上快速的免疫标记

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

生物学

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

从小鼠眼中激光捕捉高纯大梁网的基因表达分析

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

从新鲜冷冻的小鼠骨骼中产生高质量RNA的激光捕获微解剖协议

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and depth of this technology and extending it to the level of cells within the tissue is needed. Although the use of single nuclear and single cell RNA sequencing has become widespread, the expression signatures of cells obtained from tissue dissociation do not maintain spatial context. Laser microdissection (LMD) based on specific fluorescent markers would allow the isolation of specific structures and cell groups of interest with known localization, thereby enabling the acquisition of spatially-anchored transcriptomic signatures in kidney tissue. We have optimized an LMD methodology, guided by a rapid fluorescence-based stain, to isolate five distinct compartments within the human kidney and conduct subsequent RNA sequencing from valuable human kidney tissue specimens. We also present quality control parameters to enable the assessment of adequacy of the collected specimens. The workflow outlined in this manuscript shows the feasibility of this approach to isolate sub-segmental transcriptomic signatures with high confidence. The methodological approach presented here may also be applied to other tissue types with substitution of relevant antibody markers.

We describe a protocol for laser microdissection of sub-segments of the human kidney, including the glomerulus, proximal tubule, thick ascending limb, collecting duct and interstitium. The RNA is then isolated from the obtained compartments and RNA sequencing is carried out to determine changes in the transcriptomic signature within each sub-segment.

应用激光微分射发现人体肾脏组织中的区域转录学

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and ...