이 작품은 사우스 웨스턴 오클라호마 주립 대학, 웨더 확인에서 학부 학생들이 수행되었다. 이 작품은 루이 NSF - 0963258, NIH OK - INBRE 2P20RR016478, GCAT 및 OCAST HR09 - 137S에 의해 지원되었다. vsm1 (ok1468) 돌연변이가 오클라호마 진 넉아웃 컨소시엄에 의해 고립되었다. 저자는 프로젝트 수행 중에 귀중한 도움을 댄 Stefanovic와 태너 휠러 감사드립니다.

<ol>
	<li>Chai, A., Withers, J., Koh, Y. H., Parry, K., Bao, H., Zhang, B., Budnik, V., Pennetta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hVAPB,+the+causative+gene+of+a+heterogeneous+group+of+motor+neuron+diseases+in+humans,+is+functionally+interchangeable+with+its+Drosophila+homologue+DVAP-33A+at+the+Neuromuscular+Junction.">hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the Neuromuscular Junction.</a> Hum Mol Genet. 17, 266-280 (2008).</li><li>Tsuda, H., Han, S. M., Yang, Y., Tong, C., Lin, Y. Q., Mohan, K., Haueter, C., Zoghbi, A., Harati, Y., Kwan, J., Miller, M., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Amyotrophic+Lateral+Sclerosis+8+protein+VAPB+is+cleaved,+secreted,+and+acts+as+a+ligand+for+Eph+receptors.">The Amyotrophic Lateral Sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors.</a> Cell. 133, 963-977 (2008).</li><li>Dresbach, T., Torres, V., Wittenmayer, N., Altrock, W. D., Zamorano, P., Zuschratter, W., Nawrotzki, R., Ziv, N. E., Garner, C. C., Gundelfinger, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+Active+Zone+Precursor+Vesicles.">Assembly of Active Zone Precursor Vesicles.</a> Journal of Biological Chemistry. 281, 6038-6047 (2005).</li><li>Lustgarten, V., Gerst, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+VSM1+Encodes+a+v-SNARE+Binding+Protein+That+May+Act+as+a+Negative+Regulator+of+Constitutive+Exocytosis.">Yeast VSM1 Encodes a v-SNARE Binding Protein That May Act as a Negative Regulator of Constitutive Exocytosis.</a> Molecular and Cellular Biology. 19, 4480-4494 (1999).</li><li>Vi&#241;uela, A., Snoek, L., Riksen, J., Kammenga, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Gene+Expression+Analysis+in+Response+to+Organophosphorous+Pesticide+Chlorpyrifos+and+Diazinon+in+C.+elegans.">Genome-Wide Gene Expression Analysis in Response to Organophosphorous Pesticide Chlorpyrifos and Diazinon in C. elegans.</a> PloS one. 5, 1-8 (2010).</li><li>Niwa, R., Hada, K., Moliyama, K., Ohniwa, R., Tan, Y., Olsson-Carter, K., Chi, W., Reinke, V., Slack, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=elegans+sym-1+is+downstream+target+of+the+hunchback-like-1+developmental+timing+transcription+factor.">elegans sym-1 is downstream target of the hunchback-like-1 developmental timing transcription factor.</a> Cell Cycle. 8, 4147-4154 (2009).</li><li>Kershner, A., Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+mRNA+targets+for+Caenorhabditis+elegans+FBF,+a+conserved+stem+cell+regulator.">Genome-wide analysis of mRNA targets for Caenorhabditis elegans FBF, a conserved stem cell regulator.</a> PNAS. 107, 3963-3941 (2010).</li><li>Hammock, E., Eagleson, K., Barlow, S., Earls, L., Miller, D., Levitt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologs+of+genes+expressed+in+Caenorhabditis+elegans+GABAergic+neurons+are+also+found+in+the+developing+mouse+forebrain.">Homologs of genes expressed in Caenorhabditis elegans GABAergic neurons are also found in the developing mouse forebrain.</a> Neural Development. 5, 1-14 (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

본 연구에서는 다양한 생물 학적 현상을 기본 결정 유전자 발현 프로필을 사용할 수있는 쉽고 사용자 친화적인 프로토콜을 개발했습니다. 이 프로토콜에서 총 RNA가 먼저 격리와 RNA의 분리 우리의 방법은 크게 페놀 extractions를 생략하고 5,6 정리를위한 화성 수지를 사용하여 간단하게되었습니다 DNase I.를 사용하여 치료했습니다. 간단히, C. 엘레간스의 cuticule 및 세포 세포막은 액체 질소와 분자 수지로 분쇄와 냉동 nematodes에 의해 금이 - 오픈되었습니다. 다음 추출된 RNA는 cDNA 합성 전에 DNase I로 치료했다. 나중에 단계는 unspecific hybridizations을 최소화하기 위해 추가되었다; 게놈 DNA와 RNA 오염된 샘플 간섭을 소개하고 많은 거짓 - 반응을 생산 수 있습니다. 그런 다음, 야생 입력하고 vsm - 1 (ok1468) 돌연변이의 cDNAs은 Cy3와 Cy5 캡처 시퀀스 태그와 C.로 hybridized되었다 엘레간스의 microarrays은 GCAT 프로그램을 통해 얻은. 이 시스템은 유사 제품보다 더 민감하고, 그 두 색상의 디자인은 큰 시간과 비용 효율 7,8의 결과, 필요한 배열의 수를 감소시킵니다. 또한이 시스템은 다른 유사한 시스템의 전문 장비가 필요하지 않으므로,이 절차는 표준 실험실 설정 7 수행할 수 없습니다. 셋째, 형광 hybridized 배열 이미지는 오픈 소스 소프트웨어 MAGICTool를 사용하여 분석되었고, 유전자 발현 프로파일이 만들어졌습니다. MAGICTool은 학부에서 포스트 박사로, 쉽게 모든 경험 수준의 개인에 의해 활용되는 사용자 친화적인 프로그램입니다. 그러나, 최적의 성능은 대용량 메모리 가용성을 필요로합니다. 마지막으로, 후보 유전자 실시간 PCR과 검증되었습니다 microarray 분석을 사용하여 획득하였습니다. 게놈 접근 방식이 생물 학적 현상을 연구를위한 효율적인 방법에도 불구하고, 하나는 고려 소요됩니다 몇 가지 제한이있다. 인쇄 oligonucleotide가 보존 시퀀스에서 가져온 경우 첫째,이 microarray 프로토콜의 특이성에도 불구하고, 가족 내에서 성적은 서로 구별할 수 있습니다. 실시간 PCR은 유전자 가족 내에서 공통점에 대한 보상, 심지어 같은 유전자의 특정 isoforms 구별 수 있습니다. 그러나, 실시간 PCR과 동일하게 생명체의 수명에 걸쳐 표현 진정한 컨트롤을 찾기 위해 도전입니다. 이 때문에, 결과는 상대적인 것입니다. proteomic 접근은 우리의 기술을 하나의 대안입니다. 개별 단백질은 2D 겔 전기 영동과 질량 분석계로 확인을 사용하여 격리 수 있습니다. 우리의 연구는 특히 주요 정자 단백질 (MSP) 가족의 여러 구성원이 향상된 시냅스 밀도 vsm - 1 돌연변이 nematodes에서 유도되는 보여줍니다. MSPs는 C.에 고유한 엘레간스 및 oocyte의 성숙과 배란에 대한 책임이 있습니다. 차이 1 과일 파리의 신경근육학 교차점에서 presynaptic 부통 번호 및 크기의 조절 가능성이 주요 정자 단백질 도메인을 포함하는 분자에 의해 제어됩니다 것을 보여주었다. 쓰다 및 동료에 의해 ​​연구는 2 개의 주요 정자 단백질 도메인 수용체 티로신 키나제 신호 폭포를 실행, Ephrine 수용체에 대한 리간드 역할을 수 증명하고있다. 또한, 실시간 PCR 분석 확인 및 MSP 가족, MSP - 32 중 하나를 회원에게 microarray 결과를 좁혀. 함께 촬영, 여기에서 제시한 작품은 중앙 신경 과학의 딜레마의 게놈 차원의 분석뿐만 아니라 과학적 노력의 학부 연구의 파워를 나타냅니다.

<table border="1"><tbody><tr><td> 구성 요소 </td><td> 카탈로그 # </td><td> 회사 </td></tr><tr><td> RNA 나중 </td><td> AM7020 </td><td> 앰비온 </td></tr><tr><td> 분자 연삭 수지 </td><td> 786-138 </td><td> Gbiosciences </td></tr><tr><td> RNeasy 미니 키트 </td><td> 74,104 </td><td> Qiagen </td></tr><tr><td> Rnase없는 아가로 오스 LE </td><td> AM9040 </td><td> 앰비온 </td></tr><tr><td> NorthernMax Gly 10X 젤 준비 / 실행 버퍼 </td><td> 8678 </td><td> 앰비온 </td></tr><tr><td> RNA 밀레니엄 마커 </td><td> AM7150 </td><td> 앰비온 </td></tr><tr><td> Glyoxal 샘플 로딩 다이 </td><td> 8551 </td><td> 앰비온 </td></tr><tr><td> DNase 세트 </td><td> 79,254 </td><td> Qiagen </td></tr><tr><td> RNeasy MinElute 키트 </td><td> 74,204 </td><td> Qiagen </td></tr><tr><td> RT 효소와 배 반응 완충액 </td><td> RT300320 </td><td> Genisphere </td></tr><tr><td> 배열 350 </td><td> W300180 </td><td> Genisphere </td></tr><tr><td> QIAquick PCR의 정제 키트 </td><td> 28,104 </td><td> Qiagen </td></tr><tr><td> 20X SSC </td><td> 82021-4846 </td><td> VWR </td></tr><tr><td> 가지각색 연어 정자 DNA </td><td> AM9680 </td><td> 앰비온 </td></tr><tr><td> SDS 솔루션 </td><td> V6551 </td><td> Promega </td></tr><tr><td> DTT EM </td><td> 3860 </td><td> VWR </td></tr><tr><td> iScript cDNA 합성 키트 </td><td> 170-8890 </td><td> BioRad </td></tr><tr><td> IQ SYBR 그린 Supermix </td><td> 170-8880 </td><td> BioRad </td></tr></tbody></table>

determining genetic expression profiles in c. elegans using microarray and real-time pcr

시냅스는 대상 세포에 신호 세포와 postsynaptic 터미널에서 presynaptic 활성 영역으로 구성되어 있습니다. 화학 시냅스의 경우, 메시지는 presynaptic 터미널에서 발표하고 postsynaptic 수용체 세포에 신경 전달 물질에 의해 접수 실시하고 있습니다. Caenorhabditis 엘레간스에있는 우리 이전의 연구는 VSM - 1 부정 exocytosis을 조절하는 것으로 나타났습니다. 또한 vsm - 1 돌연변이에 시냅스의 분석 보여준 완벽한 기능 VSM - 1 증가 시냅스 연결을 부족한 동물. 이러한 예비 조사 결과를 바탕으로, 우리는 가상 그 C. 엘레간스 VSM - 1 synaptogenesis에서 중요한 역할을 수 있습니다. 이 가설을 테스트하려면 두 번 표시 microarray 분석이 수행되었고, 유전자 발현 프로파일이 결정됩니다. 첫째, 총 RNA는 고립되었다, reversely cDNA로 베꼈는데하고, DNA를 microarrays에 hybridized. 그런 다음, 형광 프로브 하이브 리다이 제이션의 인 silico 분석 향상된 synaptogenesis과 돌연변이의 주요 정자 단백질 가족 (MSP)의 회원 코딩 많은 유전자의 중요한 유도을 공개했다. MSPs는 C로 정자의 주요 구성 요소입니다 엘레간스 및 선충류의 oocyte의 성숙과 배란 신호를 나타납니다. 과일 파리에서 차이와 동료 1 MSP 같은 분자가 신경근육학 교차로에서 presynaptic 부통 번호와 크기를 규제 것을 보여주었다. 또한, 쓰다 및 동료에 의해 ​​수행 분석 2 MSPs는 에프 수용체와 트리거 수용체 티로신 키나제의 신호 폭포에 대한 리간드 역할을 수 있습니다 것을 제안했습니다. 마지막으로, 실시간 PCR 분석 MSP - 32에 대한 코딩 유전자가 vsm - 1 (ok1468) 돌연변이의 유발 것을 뒷받침. 함께 찍은, 우리 실험실에 의해 수행 연구 vsm - 1 돌연변이 MSP - 32 신호에 의해 중재 수 시냅스 밀도에 상당한 증가를 가지고 나타났습니다.

Watch this Scientific Journal Video about Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR at JoVE.com

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray 분석은 유전자 표현의 프로필을 결정하기 위해 실시되었다<em&gt; C. 엘레간스</em&gt; 실시간 PCR은 microarray 데이터를 확인하고 수치로 사용되었습니다.

의 유전자 표현 프로필 결정 C. 엘레간스 Microarray 및 실시간 PCR을 사용하여

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

determining-genetic-expression-profiles-c-elegans-using-microarray

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Biology

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

23.0K 조회수.  Southwestern Oklahoma State University. Microarray 분석은 유전자 표현의 프로필을 결정하기 위해 실시되었다 C. 엘레간스 실시간 PCR은 microarray 데이터를 확인하고 수치로 사용되었습니다.

비디오: Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

의 Transcriptomic 분석<em&gt; C</em&gt;.<em&gt; 간스</em갤럭시 프로젝트에 턱시도 제품군을 통해&gt; RNA 시퀀싱 데이터

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

연구

유전학

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on animal physiology and behavior. Recently, we showed that modulation of longevity in the nematode Caenorhabditis elegans by food abundance is more complex than previously recognized. The responsiveness of the lifespan to changes in food level is determined by specific genes that act by controlling information processing within a neural circuit. Our framework combines genetic analysis, high-throughput quantitative imaging and information theory. Here, we describe how these techniques can be used to characterize any gene that has a physiological relevance to broad-range dietary restriction. Specifically, this workflow is designed to reveal how a gene of interest regulates lifespan under broad-range dietary restriction; then to establish how the expression of the gene varies with food level; and finally, to provide an unbiased quantification of the amount of information conveyed by gene expression about food abundance in the environment. When several genes are examined simultaneously under the context of a neural circuit, this workflow can uncover the coding strategy employed by the circuit.

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Here, we present a framework to relate broad-range dietary restriction to gene expression and lifespan. We describe protocols for broad-range dietary restriction and for quantitative imaging of gene expression under this paradigm. We further outline computational analyses to reveal underlying information processing features of the genetic circuits involved in food-sensing.

C. 선 충 에서 광범위 한 범위 식이 제한 유전자 식 수준 동안 수명 변조에서 인코딩된 정보의 정량화

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

단일 및 벌크 C.의 고처리량 정량적 RT-PCR. 나노 유체 기술을 사용하여 샘플

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

에 Postembryonic Phenotypes을 식별하기 위해 RNAi 심사 C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

생물학

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

의 정량 및 자동화된 고속 처리량 게놈 전체의 RNAi 화면 C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (&#8805;75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation &#8804;60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (&#8805;80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

This protocol describes a rapid and low material consuming procedure for the integration of transgenic extrachromosomal arrays into the Caenorhabditis elegans genome using ultra violet (UV) irradiation. Furthermore, this protocol is particularly well suited for transgenic lines that transmit extrachromosomal arrays at a high rate.

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

결합 된 뉴클레오티드와 꼬마 선 충 의 단백질 추출

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

An Overview of Gene Expression

Gene expression is the complex process where a cell uses its genetic information to make functional products. This process is regulated at multiple stages, and any misregulation could lead to diseases such as cancer. This video highlights important historical discoveries relating to gene expression, including the understanding of how distinct combinations of DNA bases encode the amino acids that make up proteins. Key questions in the field of gene expression research are explored, followed by a discussion of several techniques used to measure gene expression and investigate its regulation. Finally, we look at how scientists are currently using these techniques to study gene expression.

Gene Expression Research: Concepts and Techniques

유전자 발현의 개요

Gene expression is the complex process where a cell uses its genetic information to make functional products. This process is regulated at multiple ...

교육

JoVE Science Education

심화생물학

Expression Profiling with Microarrays

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and nucleic acids derived from samples. Using these arrays, scientists can simultaneously evaluate the expression of thousands of genes. In addition, the expression profiles of different cells or tissue types can be compared, allowing researchers to deduce how the expression of different genes change during biological processes, and thus gain insight into how the genes may function in pathways or networks.Here, JoVE explains the principles behind microarrays. This is followed by a general protocol for performing a microarray experiment, and a brief introduction to analyzing microarray data. We end on a discussion of how scientists are currently using microarrays, for example to compare gene expression between different cell types derived from cancerous and non-cancerous tissues, to study important biological problems.

마이크로 어레이를 사용한 발현 프로파일링

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and ...

RNAi in C. elegans

RNA interference (RNAi) is a widely used technique in which double stranded RNA is exogenously introduced into an organism, causing knockdown of a target gene. In the nematode, C. elegans, RNAi is particularly easy and effective because it can be delivered simply by feeding the worms bacteria that express double stranded RNA (dsRNA) that is complementary to a gene of interest. First, this video will introduce the concept of RNA interference and explain how it causes targeted gene knockdown. Then, we will demonstrate a protocol for using RNAi in C. elegans, which includes preparation of the bacteria and RNAi worm plates, culturing of the worms, and how to assess the effects of RNAi on the worms. RNAi is frequently used to perform reverse genetic screens in order to reveal which genes are important to carry out specific biological processes. Furthermore, automated reverse genetic screens allow for the efficient knockdown and analysis of a large collection of genes. Lastly, RNAi is often used to study the development of C. elegans. Since its discovery, scientists have used RNAi to make tremendous progress on the understanding of many biological phenomena.

RNA Interference RNAi in Caenorhabditis elegans

예쁜 꼬마 선충에 RNAi의 적용

RNA interference (RNAi) is a widely used technique in which double stranded RNA is exogenously introduced into an organism, causing knockdown of a target ...