这项工作得到了国立药物滥用中心（011806 P50的DA）和美国军队国防部（W81XWH - 07 - 1 - 0183）中的一部分。

1。打印自定义的miRNA芯片<ol><li>打印芯片的幻灯片，在一所大学或公司使用的芯片的核心设施服务。芯片制造的质量是微阵列实验的成功的最关键因素之一。如果可能的话，尝试一些服务。理想的情况下，人会一次打印50-100幻灯片，和所有的幻灯片看起来相同，具有很好的分离，个别景点，。 </li><li>对于芯片的支持，我们使用的差距II涂层的幻灯片。 </li><li>对于miRNA的探针，我们使用NCode多物种的miRNA微阵列探头设置V2的。 </li><li>溶于3 × SSC在10μM所有的寡核苷酸和异体四上打印的幻灯片。根据GAAPS第二张幻灯片的说明，修复紫外线照射的幻灯片。标签幻灯片用钻石笔，以纪念该地区与印探头方。储存在22 ° C。 </li></ol> 2。样品制备<ol><li>任何方法或试剂可用于分离RNA的，只要它保留了小分子RNA。我们通常使用的Trizol（Invitrogen公司），提取总RNA，令人满意的数量和质量的RNA制剂作为一个260nm一 280nm处读数计量，。 </li><li>对于RNA标记，混合〜25μg总RNA 5&#39; - PCU - DY547 - 3“在1个缓冲区（的50 mM HEPES，pH值7.8，MgCl 2的 20毫米，10微克/ ml的BSA，10％与0.5微克二甲基亚砜）〜20单位T4 RNA连接酶1〜10毫米DTT和〜0.02-0.03终体积为10 × T4 RNA连接酶1对ATP的缓冲补充。 </li><li>允许标签上冰冰箱为2-24小时内进行。用0.3 M醋酸钠和3体积乙醇沉淀RNA。为了有效地结扎和降水超过200毫克/毫升，溶解总RNA，并保持在20μL总结扎量。 <ol><li>如果较少的RNA是，输入的核糖核酸（RNA）和5&#39; - PCU - DY547 - 3“可以缩小。/ LI&gt; <li>如果RNA是很稀，添加酵母tRNA援助降水等的载体。 </li></ol></li><li>合并和标签参考相应的DNA寡核苷酸使用的Alexa Fluor 647核酸标记试剂盒作为杂交过程控制 6 Ulysis哺乳动物的miRNA的一个子集，共有1微克。 Purify的使用CentriSep列的标记的DNA，并在避光保存在-20 ° C </li></ol> 3。芯片杂交<ol><li>首次使用幻灯片，预杂交3 × SSC，0.1％SDS，0.2％为30-60分钟BSA，过滤解决方案在幻灯片在37 ° C。淹没在水中几次，然后在异丙醇他们。干幻灯片使用离心分离机，5分钟的幻灯片在100克的适配器，在22 ° C。 </li></ol>注：我们使用康宁芯片杂交商会和伊利Lifterslips科学的mSeries进行芯片杂交。 <ol start="2"><li>冲洗李fterslips水，然后在乙醇，在空气中干燥前。放置一个芯片杂交室基地内的幻灯片，对幻灯片的顶部lifterslip。 lifterslip将覆盖探头面积，与联系幻灯片相间。 </li><li>在黑暗中，自旋向下沉淀，标记的RNA。用70％乙醇洗一次，空气干燥沉淀。颗粒应偏红。 </li><li>准备杂交溶液中含有400毫米的Na 2 HPO 4 pH值7.0，0.8％BSA，5％SDS，12％甲酰胺 6μL纯化的1/15-1/100，标记的参照DNA（2.4）每RNA样品。添加的引用DNA的数量取决于有多少种不同的寡核苷酸标记（2.4）。如果有超过100个，则需要较少的稀释。 </li><li> 〜60μL杂交液溶解RNA沉淀。 </li><li>加入20μL的水康宁芯片杂交室基地加湿井。 </li><li>添加吨他混合物标记的RNA和参照DNA的幻灯片。使用细吸管尖，轻轻触摸lifterslip边缘，允许的解决方案，进入通过吸lifterslip和幻灯片之间的空间。 </li><li>放置在基地的杂交盒盖，然后密封与金属剪辑室。 </li><li>把整个塑料袋内室，从康宁室卡带。 </li><li>将湿纸巾的容器内室的磁带（S），用保鲜膜覆盖容器内放置了37 °彗星潮湿，细胞培养孵化器〜24小时。这些注意事项（3.6，3.9，3.10），确保杂交液不干燥孵化过程中。 </li></ol> 4。杂交后处理<ol><li>逐一拆开室录音带。淹没幻灯片和其lifterslip 2 × SSC在22 ° C。 lifterslip会自然脱落的幻灯片。将其放置在0.8 × SSC。洗在0.8 × SSC幻灯片两次，然后在0.4 × SSC，总在1-2分钟的3倍。干的幻灯片，使用幻灯片适配器离心机。用清水洗净lifterslips和保存后重新使用。 </li><li>任何合适的扫描仪，如Perkin Elmer公司ScanArray 5000或分子器件的Axon GenePix 4000B芯片扫描仪扫描的幻灯片。在波长接近547 nm和647 nm的扫描，以获得相应的的图像文件。 </li><li>使用BlueFuse，如计划，量化的投入，从4.2图像文件）的像素强度。检查与程序的各个点，以排除进一步审议芯片的异常点。这些异常点通常出现杂交后处理不良的幻灯片打印或幻灯片。 </li><li>如GeneSpring和Excel进行数据分析和演示方案。总的考虑适用于微阵列数据正常化，这超出了本文的范围。 </li><li>一旦获得令人满意的信号自动对焦之三4.3），再用7地带的芯片幻灯片。在水冲洗的幻灯片，然后在预热，1毫米的NaOH和0.1 × SSC在染色盘，在62 ° C浸泡10-20分钟。重复孵化一次。洗幻灯片22几次水，轻轻摇动60分钟° C.干燥的幻灯片，使用离心机，并存储在22 ° C </li><li>要使用可再生的幻灯片，它是没有必要的预杂交再次的。只需由异丙醇水冲洗，干配种前的幻灯片。 </li></ol> 5。代表性的成果： 我们基本上遵循所描述的程序来分析全球数千样本的miRNA的表达，即在许多不同的条件下，从斑马鱼中分离人体标本的RNA。图1显示了一个非常精确和较强的杂交信号，证明对微阵列扫描图像幻灯片。皮尔逊correlati系数之间的技术复制芯片杂交〜0.99 7，说明优秀的重复性。 <img alt="图1" src="/files/ftp_upload/3250/3250fig1.jpg" /> 图1合成图像扫描的miRNA杂交后的芯片幻灯片。而黄色斑点相同的探针的DNA和RNA的杂交，导致杂交参考DNA，DY547 -标记的RNA样品的绿色斑点，红色斑点。

<ol>
	<li>Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functions+of+animal+microRNAs.">The functions of animal microRNAs.</a> Nature. 431, 350-355 (2004).</li><li>Friedman, R. C., Farh, K. K., Burge, C. B., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19, 92-105 (2009).</li><li>Chekulaeva, M., Filipowicz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+miRNA-mediated+post-transcriptional+regulation+in+animal+cells.">Mechanisms of miRNA-mediated post-transcriptional regulation in animal cells.</a> Curr. Opin. Cell Biol. 21, 452-460 (2009).</li><li>Farazi, T. A., Spitzer, J. I., Morozov, P., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNAs+in+human+cancer.">miRNAs in human cancer.</a> J. Pathol. 223, 102-115 (2011).</li><li>Small, E. M., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pervasive+roles+of+microRNAs+in+cardiovascular+biology.">Pervasive roles of microRNAs in cardiovascular biology.</a> Nature. 469, 336-342 (2011).</li><li>Thomson, J. M., Parker, J., Perou, C. M., Hammond, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+custom+microarray+platform+for+analysis+of+microRNA+gene+expression.">A custom microarray platform for analysis of microRNA gene expression.</a> Nat. Methods. 1, 47-53 (2004).</li><li>Zhang, X., Xu, W., Tan, J., Zeng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stripping+custom+microRNA+microarrays+and+the+lessons+learned+about+probe:slide+interactions.">Stripping custom microRNA microarrays and the lessons learned about probe:slide interactions.</a> Anal. Biochem. 386, 222-227 (2009).</li><li>Griffiths-Jones, S., Saini, H. K., van Dongen, S., Enright, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRBase:+tools+for+microRNA+genomics.">miRBase: tools for microRNA genomics.</a> Nucl. Acids. Res. 36, D154-D158 (2008).</li><li>Landgraf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mammalian+microRNA+expression+atlas+based+on+small+RNA+library+sequencing.">A mammalian microRNA expression atlas based on small RNA library sequencing.</a> Cell. 129, 1401-1414 (2007).</li><li>Chiang, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+microRNAs:+experimental+evaluation+of+novel+and+previously+annotated+genes.">Mammalian microRNAs: experimental evaluation of novel and previously annotated genes.</a> Genes. Dev. 24, 992-1009 (2010).</li></ol>

没有利益冲突的声明。

尽管深度测序技术的最新进展，芯片仍然是一个可行的选择，为高通量DNA和RNA分析。深度测序相比，微阵列实验比较便宜，一个典型的分子生物学实验室可以执行大部分的实验和数据分析的房子，允许灵活性和节省时间。在未来的芯片可能非常适合集中审问的基因，例如集，全部或在基因组或miRNA的转录因子的一个子集，这里提出相同的原则和程序可以用于定制微阵列实验研究此外miRNA的基因家族...

<table border="1"><tbody><tr><th>项目</th><th>供应商</th><th>目录编号</th><th>评论</th></tr><tr><td> NCode多物种的miRNA微阵列探头设置V2 </td><td> Invitrogen公司</td><td> MIRMPS201 </td><td>设计的基础上miRBase 9.0版（2006年10月）。它包含〜1140未修改，34-44 NT长寡核苷酸作为探针蠕虫，蝇，斑马鱼，小鼠，大鼠，和人类的miRNA，和内部控制探头，如snoRNAs。成熟的miRNA，因此〜44 NT的大小序列互补的miRNA探针的双峰。进行分析，可以从一个特定的基因组（S）的兴趣集中在miRNA的。 </td></tr><tr><td> Trizol法</td><td> Invitrogen公司</td><td> 15596018 </td><td>我们也用丰富，标注的小RNA的一小部分，虽然总RNA样本更快，更容易准备和量化，适合于下游应用，如基因分析。 </td></tr><tr><td> T4 RNA连接酶1 </td><td>新英格兰生物学ABS </td><td> M0204L </td><td></td></tr><tr><td> Ulysis的Alexa Fluor 647核酸标记试剂盒</td><td> Invitrogen公司</td><td> U21660 </td><td>该试剂盒或类似的产品可用于实验RNA样品或对照RNA（而不是控制DNA）以及标签。 </td></tr><tr><td> 5&#39; - PCU - DY547 - 3“ </td><td> Dharmacon </td><td>产品订制</td><td>小RNA的分数可以用同样的标记结扎。 </td></tr><tr><td> CentriSep列</td><td>普林斯顿分离</td><td> CS - 901 </td><td></td></tr><tr><td> GAPSII涂幻灯片</td><td>康宁</td><td> 40004 </td><td>也可能使用其他类型的幻灯片。 </td></tr><tr><td>微阵列杂交商会</td><td>康宁</td><td> 2551或40080 </td><td>其他种杂交室和盖玻片还应工作。使用市售的杂交机可以显著减少杂交时间，例如，1〜2小时。 </td></tr><tr><td> Lifterslips </td><td>钍ermo /伊利科学</td><td> 25X60I - M5439 - 001 - LS </td><td></td></tr><tr><td> BlueFuse </td><td> BlueGenome </td><td></td><td></td></tr><tr><td> GeneSpring </td><td>安捷伦</td><td></td><td></td></tr></tbody></table>

performing custom microrna microarray experiments

小分子RNA（miRNAs）是一个大家族〜22核苷酸（nt）长的RNA分子，在真核生物中 1广泛表达。复杂的基因组编码至少有数百个miRNA的，这主要是抑制大量的靶基因的转录后2，3的表达。 miRNA的控制范围广泛的生物过程1。此外，miRNA表达的改变已经与人类疾病，如癌症，和miRNA可作为生物标志物疾病和预后4， 5。因此，重要的是理解表达和许多不同的条件下miRNA的功能。 已采用文件miRNA表达的实时PCR技术，基因芯片和深度测序的三个主要方法。的miRNA芯片技术具有高通量的优势，一般较便宜，和大多数的实验和分析的步骤可卡尔IED在分子生物学实验室，在大多数大学，医学院校和相关的医院。在这里，我们描述了一个用于执行自定义的miRNA微阵列实验的方法。一个miRNA探针组将印上产生的miRNA微阵列玻片。 RNA是隔离的使用方法或试剂，保留小分子RNA的物种，然后用荧光染料标记。作为对照，参考相应的DNA寡核苷酸的miRNA的一个子集上，也有不同的荧光染料。参照DNA将成为展示的幻灯片和杂交质量，也将用于数据规范化。 RNA和DNA混合并杂交到芯片的幻灯片，其中包含数据库中的大多数miRNA的探针。洗涤后，幻灯片扫描获取图像，个别点强度量化。这些原始信号将得到进一步的处理和相应的miRNA表达数据分析。微安rray幻灯片可以剥离和再生，以降低芯片的成本和提高的微阵列实验的一致性。相同的原则和程序适用于其他类型的定制微阵列实验。

Watch this Scientific Journal Video about Performing Custom MicroRNA Microarray Experiments at JoVE.com

Performing Custom MicroRNA Microarray Experiments

一个简单的程序执行自定义的microRNA微阵列实验描述。这些步骤包括隔离RNA，标签的RNA和参照DNA样本，杂交到芯片，芯片扫描，量化和分析杂交信号。

执行自定义的微RNA基因芯片实验

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1.  Complex genomes encode ...

performing-custom-microrna-microarray-experiments

Research

JoVE Journal

Biology

19.4K 次观看.  University of Minnesota. 一个简单的程序执行自定义的microRNA微阵列实验描述。这些步骤包括隔离RNA，标签的RNA和参照DNA样本，杂交到芯片，芯片扫描，量化和分析杂交信号。

视频: Performing Custom MicroRNA Microarray Experiments

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Gibberella zeae子囊孢子生产和收集微阵列实验。

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

科研

生物学

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

电生理实验人工双层膜的制备

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Processing the Loblolly Pine PtGen2 cDNA Microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem.1,2 PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously.3,4 Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing. 
PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips&trade; are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48&deg;C in high humidity conditions. After hybridization, a series of standard washes are done at 53&deg;C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.

The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.

处理火炬松PtGen2差异表达基因的cDNA微阵列

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

全球基因表达分析，使用斑马鱼的寡核苷酸芯片平台

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

微阵列分析FOR酿酒酵母</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

基因组编辑与的CompoZr自锌指核酸酶（ZFNs）

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and cardiovascular disease, have been shown to activate tissue and/or cell-specific miRNA expression associated with disease progression. The inhibition of miRNA expression offers the potential for a therapeutic intervention. However, traditional approaches to inhibit miRNAs, employing antagomir oligonucleotides, affect specific miRNA functions upon global delivery. Herein, we present a protocol for the in vivo cardio-specific inhibition of the miR-181 family in a rat model. A miRNA-sponge construct is designed to include 10 repeated anti-miR-181 binding sequences. The cardio-specific &#945;-MHC promoter is cloned into the pEGFP backbone to drive the cardio-specific miR-181 miRNA-sponge expression. To create a stable cell line expressing the miR-181-sponge, myoblast H9c2 cells are transfected with the &#945;-MHC-EGFP-miR-181-sponge construct and sorted by fluorescence-activated cell sorting (FACs) into GFP positive H9c2 cells which are cultured with neomycin (G418). Following stable growth in neomycin, monoclonal cell populations are established by additional FACs and single cell cloning. The resulting myoblast H9c2-miR-181-sponge-GFP cells exhibit a loss of function of miR-181 family members as assessed through the increased expression of miR-181 target proteins and compared to H9c2 cells expressing a scramble non-functional sponge. In addition, we develop a nanovector for the systemic delivery of the miR-181-sponge construct by complexing positively charged liposomal nanoparticles and negatively charged miR-181-sponge plasmids. In vivo imaging of GFP reveals that multiple tail vein injections of a nanovector over a three-week period are able to promote a significant expression of the miR-181-sponge in a cardio-specific manner. Importantly, a loss of miR-181 function is observed in the heart tissue but not in the kidney or the liver. The miRNA-sponge is a powerful method to inhibit tissue-specific miRNA expression. Driving the miRNA-sponge expression from a tissue-specific promoter provides specificity for the miRNA inhibition, which can be confined to a targeted organ or tissue. Furthermore, combining nanovector and miRNA-sponge technologies permits an effective delivery and tissue-specific miRNA inhibition in vivo.

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

Tissue-specific microRNA inhibition is a technology that is underdeveloped in the microRNA field. Herein, we describe a protocol to successfully inhibit the miR-181 microRNA family in myoblast cells from the heart. Nanovector technology is used to deliver a microRNA sponge that demonstrates significant in vivo cardio-specific miR-181 family inhibition.

体内Nanovector 心脏特异性 MicroRNA 海绵的交付

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and ...

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30%&#160;of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic.
The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment.
The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.

This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single experiment.

用于微RNA簇网络分析的CRISPR基因编辑工具

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published ...