Name: depletion of ribosomal rna for mosquito gut metagenomic rna-seq
Uploaded: 2013-04-07T14:00:00
Description: Watch this Scientific Journal Video about Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq at JoVE.com

这项工作是由NIH授予1SC2GM092789，-01A1，MS新墨西哥州立大学霍华德·休斯医学院校本科研究项目的研究学者。录影被指挥和生产由Amy Lanasa和协调的菲利普·刘易斯博士在新墨西哥州立大学的创意媒体学院。

程序 1。蚊子饲养<ol><li>后蚊子冈比亚按蚊 G3在养虫株为27.5°C，湿度80％，光/暗周期12:12小时。 </li><li>与啤酒酵母的比例为1:1与地面的猫粮喂养幼虫。 </li><li>对小鼠血后第3天出现产蛋饲料成蚊。 </li></ol> 2。蚊子肠道解剖<ol><li>高压灭菌器的清扫工具（幻灯片和钳）。 </li><li>使用抽气收集50个蚊子，把它们放在CO 2流床（终极Flypad）。冲洗顺序?...

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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+simple+method+to+enrich+mRNA+from+total+prokaryotic+RNA.">A simple method to enrich mRNA from total prokaryotic RNA.</a> Mol. Biotechnol. 10, 83-85 (1998).</li><li>Gusmao, D. S., 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=Culture-dependent+and+culture-independent+characterization+of+microorganisms+associated+with+Aedes+aegypti+(Diptera:+Culicidae)+(L.)+and+dynamics+of+bacterial+colonization+in+the+midgut.">Culture-dependent and culture-independent characterization of microorganisms associated with Aedes aegypti (Diptera: Culicidae) (L.) and dynamics of bacterial colonization in the midgut.</a> Acta Trop. 115, 275-281 (2010).</li><li>Lindh, J. 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一个复杂的微生物群落居住在蚊子的肠道生态系统的14,16,17。 Metatranscriptomic测序（RNA-seq的）上下文相关的功能信息可以透露讯问的整个微生物的转录4,18。从技术上讲，低聚的d（T）介导的富集的原核mRNA的是不适用的，由于缺乏稳定的聚（A）尾的使者。或者，rRNA的耗尽已用于mRNA的富集。在这里，我们开发了消耗rRNA基因rRNA基因探针，从自己在蚊子的肠道微生物群落宏基因组DNA一?...

新一代测序技术已经极大地推进了宏基因组学研究评估的分类组成和遗传功能的微生物组合。 RNA测序（RNA-Seq的）可以绕过文化为基础的方法，，调查微生物metatranscriptomes在不同的情况下2-5。微生物的RNA-seq的一个主要障碍是丰富表达的困难，没有稳定的聚腺苷酸化的原核细胞的mRNA分子。因此，寡d（T）介导的信使富集是不适用的。删除丰富的rRNA基因是一种替代方法来丰富表达。耗竭商业rRNA基因包，如细菌的mRNA Microexpress富集试剂盒（Ambion），RiboMinus转录分离试剂盒（细菌）（Life Technologies公司），和的mRNA-ONLY原核mRNA分离试剂盒（震中）优先降解与核酸外切酶的rRNA基因，已经使用了除去rRNA的6-8。然而，捕获探针Microexpress或RiboMinus用于除去已知的rRNA从典型的革兰氏阳性菌和革兰氏阴性菌（参见制造商的规格）是良好的，但不兼容从未知的微生物与rRNA。因此，去除效率可能较低宏基因组样品8-10。此外，mRNA丰度的保真度是有问题的核酸外切酶处理时，适用11。总体而言，基于减法rRNA基因消耗较少偏见的和更有效的宏基因组的基因富集设置10-13。 蚊子肠道容纳一个充满活力的微生物群落14。我们有兴趣在蚊子的肠道微生物利用RNA-Seq的功能特征。在一个分离的RNA样品由蚊子胆量，蚊子和微生物的RNA存在。在这里，我们描述了修改后的协议，使用社区的具体rRNA探针能够有效地消耗蚊子和微生物的rRNA基因消减杂交技术。所得mRNARNA-Seq的丰富的样本是适当的。在图1中示出的整体的工作流。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material </td>
 </tr>
 <tr>
 <td>Meta-G-Nome 				DNA isolation kit</td>
 <td>Epicentre</td>
 <td>MGN0910</td>
 <td>Metagenomic 				DNA isolation</td>
 </tr>
 <tr>
 <td>TriPure </td>
 <td>Roche</td>
 <td>11667165001</td>
 <td>Mdetagenomic 				RNA isolation</td>
 </tr>
 <tr>
 <td>MEGAscript 				T7 kit</td>
 <td>Ambion</td>
 <td>AM1334</td>
 <td>In 				vitro synthesis of RNA probes</td>
 </tr>
 <tr>
 <td>RNaseZap</td>
 <td>Ambion</td>
 <td>AM9780</td>
 <td>RNase 				free working area </td>
 </tr>
 <tr>
 <td>Biotin-16-UTP</td>
 <td>Roche</td>
 <td>11388908910</td>
 <td>In 				vitro synthesis of RNA</td>
 </tr>
 <tr>
 <td>Biotin-11-CTP</td>
 <td>Roche</td>
 <td>4739205001</td>
 <td>In 				vitro synthesis of RNA</td>
 </tr>
 <tr>
 <td>Streptavidin 				magnetic beads</td>
 <td>NEB</td>
 <td>S1420S</td>
 <td>Capture 				of rRNA hybrids</td>
 </tr>
 <tr>
 <td>Magnetic 				separation rack</td>
 <td>NEB</td>
 <td>S1506S</td>
 <td>Capture 				of rRNA hybrids</td>
 </tr>
 <tr>
 <td>RNeasy 				mini kit</td>
 <td>QIAGEN</td>
 <td>74104</td>
 <td>Purification 				of subtracted RNA</td>
 </tr>
 <tr>
 <td>RNase-Free 				DNase Set</td>
 <td>QIAGEN</td>
 <td>79254</td>
 <td>Removal 				DNA contamination </td>
 </tr>
 <tr>
 <td>Agilent 				RNA 6000 Pico Kit</td>
 <td>Agilent 				Technologies Inc.</td>
 <td>5067-1513</td>
 <td>Electropherogram 				of RNA</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Bio-Gen 				PRO200 				Homogenizer</td>
 <td>PRO 				Scientific</td>
 <td>01-01200</td>
 <td>Mosquito 				gut tissue homogenization</td>
 </tr>
 <tr>
 <td>NanoDrop 				1000 Spectrophotometer</td>
 <td>Thermo 				Scientific</td>
 <td></td>
 <td>DNA 				&amp; RNA quantitation</td>
 </tr>
 <tr>
 <td>2100 				Bioanalyzer</td>
 <td>Agilent 				Technologies Inc.</td>
 <td>G2940CA</td>
 <td>Electropherogram 				of RNA</td>
 </tr>
 </tbody>
</table>

depletion of ribosomal rna for mosquito gut metagenomic rna-seq

蚊子肠道适应动态的微生物群落昆虫的生命周期的不同阶段之间。表征的遗传能力和功能的肠道社区将提供洞察肠道菌群的影响蚊子的生命特质。宏基因组RNA序列分析转录从各种微生物，微生物群落，已经成为一个重要的工具。信使RNA通常仅包括1-3％的总RNA，而rRNA基因构成约90％。它是具有挑战性的丰富信使RNA从宏基因组微生物的RNA样品的原核表达，因为大多数物种缺乏稳定的poly（A）尾巴。这可以防止寡聚d（T）介导的mRNA隔离。在这里，我们描述了一个协议，采用衍生rRNA基因的捕获探针删除rRNA基因从宏基因组总RNA样品的样品。首先，蚊子和社区从宏基因组DNA样品的微生物和大亚基rRNA基因片段的扩增。然后，在交流了nity特定的生物素化的反义核糖体RNA的探针使用T7 RNA聚合酶在体外合成。的生物素化的rRNA基因探针杂交的总RNA。杂交捕获由链霉亲和素包被的磁珠，从总RNA中移除。基于此减法协议有效地消除蚊子和从总RNA样品中的微生物的rRNA。 mRNA的富集样品被进一步处理的RNA扩增和RNA序列。

该协议包括3个部分：（1）准备rRNA基因PCR宏基因组DNA模板（2）消减杂交技术创造特定的样品采集rRNA探针（3）消耗的rRNA总RNA。高品质的宏基因组DNA和RNA分离的整个过程是必不可少的。修改后的Meta-G-的诺姆DNA隔离协议产生高品质的宏基因组DNA从蚊子的胆量， 如图2所示。它可以是具有挑战性的蚊子胆中分离出高品质的总RNA。这可能是由于存在的各种酶，核糖核酸酶，在蚊子的胆量。我?...

Watch this Scientific Journal Video about Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq at JoVE.com

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

核糖体RNA（rRNA）的消耗协议的开发是为了丰富信使RNA（mRNA），RNA-seq中蚊子的肠道metatranscriptome的。由蚊子和其肠道微生物样品的具体rRNA探针，用于去除rRNA基因通过减法，创建了。协议的性能的影响，可能会导致在约90-99％的rRNA基因的去除。

核糖体RNA的蚊子肠道宏基因组RNA-seq的枯竭

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

Research

JoVE Journal

Biology

Large-Scale Screens of Metagenomic Libraries

宏基因组库的大型屏幕

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

科研

生物学

Protocol for Mosquito Rearing (A. gambiae)

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蚊子养育的议定书“（冈比亚按蚊）

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

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在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

CRISPR / CAS系统内切酶的底物生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells

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的16S核糖体RNA基因的扩增子的新一代测序

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

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实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，<em&gt;白纹伊蚊</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

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生理录音和黄热病蚊子的味觉附属物RNA测序<em&gt;埃及伊蚊</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

壳聚糖/干扰RNA纳米介导的基因沉默的疾病病媒蚊幼虫

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

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Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...