Name: laboratory protocol for genetic gut content analyses of aquatic macroinvertebrates using group-specific rdna primers
Uploaded: 2017-10-05T12:55:00
Description: Watch this Scientific Journal Video about Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers at JoVE.com

我们感谢 Silke 克拉森的生态系统分析和评估研究所 (创), RWTH 亚琛为她的帮助建立了该协议中使用的特定于组的入门集。我们还要感谢基尔大学的彼得马丁和劳拉 Schynawa 的合作, 在水螨的实验。我们还感谢技术实验室助理保持实验室运行顺畅, 并准备注册的公司和目录编号。这项工作得到了德国研究基金会 (DFG; 项目 GE 2219/3-1) 的支持。

1. 收集和 #160; 从田间无脊椎动物, 并准备样本进行遗传肠道含量分析. 
<ol> <li> 在每个站点收集无脊椎动物, 将个人感兴趣的个体分类为单管, 并将其直接冷冻在液氮或干冰中, 以防止肠道间隙和 DNA 降解. 
	注意: 避免在酒精中储存样品, 因为一些无脊椎动物倾向于在酒精杀死它们之前反刍. </li>
	<li> 仅使用中等大小的胃肠道 (大约和 #62; 3 mm) 动物种用于遗传肠道含量的分析因此, 样品的剩...

<ol>
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在这里, 我们提出了一个简单和经济高效的方法, pcr 确定的捕食-猎物的相互作用, 从田间样品通过组特定引物的 rDNA 地区, 可以结合其他 non-molecular 分析相同的标本。但是, 该协议中有几个关键步骤。首先, 保证所用底漆的特殊性是必不可少的。其次, 在胃肠道的制备和随后的 DNA 提取过程中, 避免肠道内容物质的污染和丢失是至关重要的。虽然 cross-sample 污染可能导致对猎物 DNA 的假阳性检测, 但肠?...

捕食-食饵的相互作用, 构成了大多数的营养相互作用和食物网络动力学, 是关键的方面, 以表征物质和能量的通量在整个食物链内和之间的生态系统, 这是生态学的主要目标之一1. 碳和养分的来源和流动的确定是进一步了解生态系统之间的连通性2。然而, 生态系统, 如河流及其汇水区, 不仅与有机物和养分的通量有关, 而且还由生物体的运动3。因此, 生境的改变中断了与这些系统挂钩的资源流动, 这不仅直接而且间接地改变了捕食者-猎物群落的组成, 从而极大地改变了这两个生态系的食物网。例如, 食物网的变化已经被证明与单一捕食物种 (例如, 虹鳟鱼)4的移动有关。这种变化可能威胁生物多样性和水生生态系统的运作。因此, 分析这一领域中的捕食者与猎物的相互作用, 对于确定人类引起的环境变化 (如水管理做法) 对水生生态系统当地生物多样性的影响至关重要。
由于在复杂的系统中跟踪营养关联是困难的, 因此已经建立了几种方法来评估在5字段中的喂养交互。传统上, 在田间觅食相互作用的研究是基于对猎物遗骸的视觉识别在解剖内脏和需要广泛的知识关于形态学猎物多样性。视觉肠内容分析提供了一些消费者群体的资源使用的洞察力 (例如,龙虾的饮食的季节性变化6和鱼7,8或喂养偏好的桡足类)。然而, 消化的物理过程使视觉肠内容分析变得困难, 通常会漏掉软体的猎物-有机体9。对于以液体摄取为食的物种或某些无脊椎动物的消费者, 在进食前密集粉碎他们的食物, 如片, 在肠道内容中对猎物种类的视觉识别是不可能的10。由于这些限制, 分子分析提供了一个有希望的选择。
分子分析现在已经成为一种常见的工具, 允许快速和精确的猎物检测肠道内容。这种技术的范围多种多样: 基于单克隆抗体或聚合酶链反应 (PCR) 的策略经常被使用, 因为它们具有高特异性和敏感性11。开发新的单克隆抗体是时间和成本密集型, 因此, 当抗体不存在11时, 其他分子技术的应用就更有用了。另一种常见的方法是放大脱氧核糖核酸 (脱氧核糖核酸) 的区域, 象核糖体核糖核酸 (RNA) 基因存在于多数种类, 使用普遍引物设置12,13。在使用这种技术时, 通常不可能在混合来源的 DNA14中确定整个捕食生物体的范围。有效的方法, 以避免这种缺点是使用组特定的入门集的遗传肠道内容分析。设计为只放大特定目标群的 DNA 区域并排除所有其他物种15,16, 组特定的引物可在指定组的分类级别上识别捕食生物体, 而无需时间和成本密集型的二次分析。然而, 像所有的肠道内容分析, 这种分析只提供了一个喂养行为的快照。因此, 结合分子肠含量分析与时间自然示踪剂 (例如,稳定同位素) 的分析被认为是有益的1,2。
在这里, 我们描述了一个详细的方法, pcr 调查的捕食-猎物的相互作用使用组特定的引物的核核糖体 DNA (rDNA) 地区与稳定同位素分析的同一标本。我们描述了通过琼脂糖凝胶电泳检测单个猎物群的 DNA。此外, 我们还提供了一个机会, 进一步下游分析 PCR 产品的这种特定的群体特异性底漆适用于任何时候更高的分类分辨率比底漆的特异性是必需的。由于单股 DNA (ssDNA) 片段形成了由其主序17确定的三级构象, 因此由这种基团特异的引物放大的片段的微小变化会导致构象变化。这种变化可以检测到单链构象多态性 (SSCP) 分析与聚丙烯酰胺凝胶17,18, 使更精确的识别猎物生物体 (下至物种水平)。
虽然琼脂糖凝胶电泳是一个常见的和廉价的工具, 可视化 dna 片段和确定其近似长度19, 分辨率取决于 dna 的数量和染色染料使用20。通常, 在使用纯 DNA 样本时, 可视化是直接的, 但是在消费者的肠道中潜在的少量的猎物 dna 可能会使琼脂糖凝胶电泳结果的评分复杂化。尽管如此, 这种检测方法是可行的, 以筛选少量的消费样本从外地的一个或几个猎物群, 但复杂的评分, 使筛选的大量样本的多个猎物群非常时间密集, 从而不切实际.更灵敏的检测方法是通过毛细管电泳自动分析碎片, 另外还允许确定片段的确切长度21。一些微卫星研究表明, 通过使用不同的荧光染料作为标签, 可以通过自动片段分析22,23来检测和确定可比较长度的不同片段. 24。因此, 我们还提出了一个详细的协议, 并行检测 DNA 从多个猎物群使用 PCR 与标记组特定的底漆集和检测通过自动片段分析与自动排序器。此外, 我们提出的案例研究的结果表明, 通过自动片段分析检测猎物的 DNA 是一种方法, 也使相对量化的摄入猎物。

<table>
	<tbody>
		<tr>
			<td>liquid nitrogen</td>
			<td>Any</td>
			<td>NA</td>
			<td>For transport of field samples of macroinvertebrates to the laboratory.</td>
		</tr>
		<tr>
			<td>1.5 ml reaction tubes - Safelock</td>
			<td>Any</td>
			<td>NA</td>
			<td>For collection of consumer specimens in the field and subsequent conservation in liquid nitrogen reaction tubes do not necessarily need to be PCR clean. To avoid exploding of the tubes, the lid of the tubes should be punctured with a needle.</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Any</td>
			<td>NA</td>
			<td>Make sure magnification is suitable to prepare the gastro-intestinaltract of the size of consumer of interest.</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Any</td>
			<td>NA</td>
			<td>To dissect the gastro-intestinal tract of consumer specimens under the stereomikroscope.</td>
		</tr>
		<tr>
			<td>Stainless steel foceps: Dumont forceps No. 7</td>
			<td>Bioform</td>
			<td>B40d</td>
			<td>This curved stainless steel forceps can sometimes be very helful to hold specimen fixed inbetween the curved part without puncturing the gastro-intestinal tract.</td>
		</tr>
		<tr>
			<td>Stainless steel foceps: Dumont forceps Electronic No. 5</td>
			<td>Bioform</td>
			<td>B40b</td>
			<td>This fine stainless steel forceps is ideally suited for the preparation of the gastro-intestinal tract of consumer specimens.</td>
		</tr>
		<tr>
			<td>DNA-ExitusPlus</td>
			<td>AppliChem</td>
			<td>A7409,1000RF</td>
			<td>Decontamination solution for the removal of DNA and RNA contaminations</td>
		</tr>
		<tr>
			<td>fuzz-free paper towel</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab scale</td>
			<td>Any</td>
			<td>NA</td>
			<td>Precision needs to be at least 0.01 g.</td>
		</tr>
		<tr>
			<td>Microlitre pipettes (0.5-10/10-100/50-200/100-1000 &#956;L)</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Tubes 2.0 ml</td>
			<td>VWR</td>
			<td>211-2165</td>
			<td>It is crucial that these raction tubes are free of DNA, DNase, RNase and PCR inhibitors (PCR clean)!</td>
		</tr>
		<tr>
			<td>Safe-Lock Tubes 1.5 ml</td>
			<td>VWR</td>
			<td>211-2164</td>
			<td>It is crucial that these raction tubes are free of DNA, DNase, RNase and PCR inhibitors (PCR clean)!</td>
		</tr>
		<tr>
			<td>Sodium chloride &#8805;99.5%, p.a., ACS, ISO</td>
			<td>carlroth</td>
			<td>3957.3</td>
			<td>For salt extraction buffer (SEB) and 5M NaCl-solution required for extraction of DNA.</td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)-aminomethane Tris</td>
			<td>carlroth</td>
			<td>4855.2</td>
			<td>For salt extraction buffer (SEB) and TBE buffer</td>
		</tr>
		<tr>
			<td>Microlitre pipettes (0.5-10/10-100/50-200/100-1000 &#956;L)</td>
			<td>carlroth</td>
			<td>8043.1</td>
			<td>For salt extraction buffer (SEB) and TBE buffer</td>
		</tr>
		<tr>
			<td>Boric acid &#8805;99.8 %, p.a., ACS, ISO</td>
			<td>carlroth</td>
			<td>6943.2</td>
			<td>For TBE buffer. Not needed if commercialy available TBE buffer is obtained.</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>carlroth</td>
			<td>4360.1</td>
			<td>For extraction of DNA</td>
		</tr>
		<tr>
			<td>Proteinase K lyophil.</td>
			<td>carlroth</td>
			<td>7528.1</td>
			<td>Prepare a working solution containing 10 mg/ml.</td>
		</tr>
		<tr>
			<td>TissueLyser II</td>
			<td>Quiagen</td>
			<td>85300</td>
			<td>Bead mill for homogenization of the samples.</td>
		</tr>
		<tr>
			<td>Stainless Steel Beads, 5 mm</td>
			<td>Quiagen</td>
			<td>69989</td>
			<td>For homogenization of samples.</td>
		</tr>
		<tr>
			<td>Heating Thermoshaker</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge: himac CT 15 RE</td>
			<td>Hitachi</td>
			<td>NA</td>
			<td>Refrigerated centrifuge used for centrifugation during the extraction of DNA (Protocol 1).</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>carlroth</td>
			<td>6752.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 99.8 %, p.a.</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Molecular biology grade (ddH2O)</td>
			<td>AppliChem</td>
			<td>A7398,1000</td>
			<td>nuclease-free (stream sterilized and DEPC treated) water used for re-suspension of DNA and whenever refered to ddH2O within the manuscript</td>
		</tr>
		<tr>
			<td>Unmodified Olegonucleotides</td>
			<td>Eurofins MWG Operon</td>
			<td>NA</td>
			<td>Primers unlabeled</td>
		</tr>
		<tr>
			<td>Modified olegonucleotides</td>
			<td>Metabion International AG</td>
			<td>NA</td>
			<td>Primers labelled</td>
		</tr>
		<tr>
			<td>peqGOLD Taq all inclusive</td>
			<td>VWR Peqlab</td>
			<td>01-1000</td>
			<td>Taq all inclusive for PCR reaction mixtures (reaction buffer Y and S, dNTPs and MgCl2)</td>
		</tr>
		<tr>
			<td>Primus 96 advanced gradient thermocycler or peqStar96x Universal Gradient</td>
			<td>Peqlab</td>
			<td>NA</td>
			<td>Primer sets were originally established on a Primus 96 advanced gradient thermocycler. As this cycler was quite old, we than checked if our primer sets are efficient and specific under the same conditions using the peqSta96x (which also could simulate the older model). However, tests showed that our primers work well and are still specific (see Koester et al. 2013) without using the simulation of the old machine.</td>
		</tr>
		<tr>
			<td>Standard 96 Well PCR Plates</td>
			<td>VWR Peqlab</td>
			<td>732-2879</td>
			<td>Used for PCR reactions.</td>
		</tr>
		<tr>
			<td>Low Profile Tube Stripes 0.15 ml with Flat Cap Stripes</td>
			<td>VWR Peqlab</td>
			<td>732-3223</td>
			<td>Cap stripes used to close the PCR plates.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Peqlab</td>
			<td>35-1020</td>
			<td>Used for gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical flask</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Measuring cylinder</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal electrophoresis unit and respective combs with teeth</td>
			<td>Any</td>
			<td>NA</td>
			<td>For agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Electrophoresis Power Supply PS3002</td>
			<td>Life Technologies</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide solution 1 %</td>
			<td>carlroth</td>
			<td>2218.1</td>
			<td>Used to prepare staining bath for agarose gels.</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>carlroth</td>
			<td>6749.1</td>
			<td>Needded for the denaturation buffer for SSCP analyses.</td>
		</tr>
		<tr>
			<td>Bromphenol blue</td>
			<td>AppliChem</td>
			<td>A3640.0010</td>
			<td>Needed for the loading dye for agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>carlroth</td>
			<td>4621.1</td>
			<td>Needed for the loading dye for agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>100 bp-DNA-Ladder extended</td>
			<td>carlroth</td>
			<td>T835.1</td>
			<td>As size standard for agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>UV-Documentation system</td>
			<td>Any</td>
			<td>NA</td>
			<td>To visualize gel electrophoresis results.</td>
		</tr>
		<tr>
			<td>Rotiphorese NF-Acrylamide/Bis-solution 40 % (29:1)</td>
			<td>carlroth</td>
			<td>A121.1</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>Ammonium peroxydisulphate</td>
			<td>carlroth</td>
			<td>9592.2</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>foldback clips (32 mm)</td>
			<td>Any</td>
			<td>NA</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>square-shaped plastic box (21 &#215; 6.5 &#215; 5 cm)</td>
			<td>Any</td>
			<td>NA</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>carlroth</td>
			<td>23.67.1</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>RC 10 Digital chiller</td>
			<td>VWR</td>
			<td>462-0138</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Vertical electrophoresis unit P10DS (OWL Separation Systems)</td>
			<td>VWR</td>
			<td>700-2361</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Notched glass plate</td>
			<td>VWR</td>
			<td>730-0261</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Blank glass plate</td>
			<td>VWR</td>
			<td>730-0260</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Spacers</td>
			<td>VWR</td>
			<td>730-0262</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Comb with 25 teeth</td>
			<td>VWR</td>
			<td>730-0294</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Shaking platform</td>
			<td>Any</td>
			<td>NA</td>
			<td>For constant shaking of the staining bath.</td>
		</tr>
		<tr>
			<td>GelStar Nucleic Acid Stain (Lonza Rockland, Inc.)</td>
			<td>Biozym</td>
			<td>850535 (50535)</td>
			<td>For staining of polyacrylamide gels.</td>
		</tr>
		<tr>
			<td>GenomeLab DNA Size Standard Kit 400bp</td>
			<td>AB Sciex</td>
			<td>608098</td>
			<td>For automated fragment analyses. Size stanard for Batch B.</td>
		</tr>
		<tr>
			<td>GenomeLab DNA Size Standard Kit 600bp</td>
			<td>AB Sciex</td>
			<td>608095</td>
			<td>For automated fragment analyses. Size stanard for Batch A.</td>
		</tr>
		<tr>
			<td>Sample Loading Solution (SLS)</td>
			<td>AB Sciex</td>
			<td>608082</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Sequenzing plate - 96 Well Polypropylene PCR Microplate</td>
			<td>Costar</td>
			<td>6551</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Plate centrifuge, PerfectSpin P</td>
			<td>VWR Peqlab</td>
			<td>NA</td>
			<td>mini plate centrifuge used to spin down PCR products.</td>
		</tr>
		<tr>
			<td>Buffer plate - 96 Well EIA/RIA Clear Flat Bottom Polystyrene Not Treated Microplate</td>
			<td>Corning</td>
			<td>9017</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>GenomeLab Seperation buffer</td>
			<td>AB Sciex</td>
			<td>608012</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Separation Gel - LPAI</td>
			<td>AB Sciex</td>
			<td>608010</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Sequenzer - CEQ8000 Genetic Analysis System</td>
			<td>Beckman Coulter</td>
			<td>NA</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>GeneMarker V1.95</td>
			<td>Softgenetics</td>
			<td>NA</td>
			<td>To analyze results of automated fragment analyses.</td>
		</tr>
	</tbody>
</table>

laboratory protocol for genetic gut content analyses of aquatic macroinvertebrates using group-specific rdna primers

分析食物网对于更好地了解生态系统是至关重要的。例如, 食物网络的相互作用可能会因非土著物种的入侵而发生严重的变化。然而, 在许多情况下, 对场捕食者-食饵相互作用的精确识别是困难的。这些分析通常是基于对肠道内容的视觉评估或稳定同位素比率的分析 (δ15N 和δ13C)。这些方法需要对个别捕食生物体的形态多样性或同位素特征进行全面的了解, 从而导致对猎物生物体的准确识别的障碍。视觉肠道内容分析, 特别是低估了软体的猎物有机体, 因为浸泡, 摄食和消化的猎物有机体, 使鉴定的特定物种很难。因此, 聚合酶链反应 (PCR) 为基础的策略, 例如使用组特定的入门集, 提供了一个强大的工具, 调查食物网络互动。在这里, 我们描述了详细的协议, 以调查的肠道内容的动物消费者从现场使用的组特定的入门套核核糖体脱氧核糖核酸 (rDNA)。DNA 可以从整个标本中提取 (在小分类群的情况下), 或者从野外采集的标本的肠道内容。dna 模板的存在和功能的效率需要直接从被测试的个体中得到确认, 使用针对各自 dna 亚基的通用引物集。我们还证明, 被消耗的猎物可以进一步确定的物种水平通过 PCR 与未修改的组特异性引物结合随后单链构象多态性 (SSCP) 分析使用聚丙烯酰胺凝胶。此外, 我们还表明, 使用不同的荧光染料作为标签, 可以通过自动片段分析从多个肠道含量样本中对不同猎物群的 DNA 片段进行平行筛选。

我们成功地使用了本协议中描述的步骤进行不同研究中的饮食分析。在本节中, 我们将介绍该协议不同部分的适用性的示例。
以 SSCP 分析为例, 演示了作者28建立的组特定引物集的有效性以及对 PCR 产物进行进一步下游分析的潜力, 并进行了饲喂试验。个体的Dikerogammarus villosus作为捕食者和Caenis的?...

Watch this Scientific Journal Video about Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers at JoVE.com

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

无脊椎动物最常见的肠道成分分析是可视的。由于需要对猎物生物体的形态多样性有强烈的了解, 他们会想念身体柔软的猎物, 由于对猎物的强烈粉碎, 对包括片在内的一些生物体来说几乎是不可能的。我们提供了详细的, 新的遗传方法, 动物猎物识别在饮食中的片。

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

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生物学

Double Whole Mount in situ Hybridization of Early Chick Embryos

双整装原位杂交技术在早期鸡胚

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

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

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

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

人类心肌细胞的生成：从馈线人类诱导多能干细胞的分化协议

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

DNA Electroporation, Isolation and Imaging of Myofibers

DNA电穿孔，肌纤维的分离和成像

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Cellular Redox Profiling Using High-content Microscopy

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, ...