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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JoVE Journal

Biology

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

22.9K 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

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Microinjection을 사용하여 안정적인 트렌스​​ 제닉 C. 엘레간스의 생성

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

연구

생물학

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.

Zebrafish Oligonucleotide Microarray의 플랫폼을 사용하여 글로벌 유전자 발현 분석

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

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

써모 과학 솔라리스 qPCR 어세를 사용하여 정량 실시간 PCR

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

에메랄드 애쉬 보러에서 유전자 발현 분석 ( Agrilus planipennis)를 사용하여 정량 리얼 타임 PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

정량 실시간 PCR (qPCR) 배열을 사용하여 사전 마이크로 RNAS과 microRNAs의 프로 파일링

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

에서 지방 질량의 생화학 및 높은 처리량 미세 평가<em&gt; Caenorhabditis Elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

항체 염색법에<em&gt; C. 엘레</em&gt; &quot;동결 크래킹 사용&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify&#160;unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3&#8217;- and 5&#8217;- sequences adjacent to the integrated lentiviral vector.

Linear Amplification Mediated PCR &amp;#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear-amplification mediated (LAM)-PCR is a method developed to identify the exact positions of integrating viral vectors in the genome. The technique has evolved to be the superior method to study clonal dynamics in gene therapy patients, biosafety of novel vector technologies, T-cell diversity, cancer stem cell models, etc.

선형 증폭 매개 PCR - 알 수없는 측면에서는 DNA의 유전 적 요소와 특성의 현지화

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

프로브 기반의 실시간 PCR은 마이크로 RNA의 정량 측정을위한 접근 방법

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag 분석 - 높은 처리량, Sc2.0 유전형에 대한 실시간 PCR 분석

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...