Name: performing custom microrna microarray experiments
Uploaded: 2011-10-28T12:00:00
Description: Watch this Scientific Journal Video about Performing Custom MicroRNA Microarray Experiments at JoVE.com

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...

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	<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>

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 ...

Research

JoVE Journal

Biology

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

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

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

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

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

Microarray Analysis for Saccharomyces cerevisiae

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 with CompoZr Custom Zinc Finger Nucleases ZFNs

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

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

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 ...

Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

利用定制设计的趋电钱伯斯研究前列腺细胞定向迁移

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and ...

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

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

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 ...