Este trabalho foi realizado pelos alunos de graduação em Southwestern Oklahoma State University, OK Weatherford. Este trabalho foi financiado pela NSF RUI-0963258, NIH OK-INBRE 2P20RR016478, GCAT e OCAST HR09-137S. O vsm1 (ok1468) mutantes foi isolado pelo Consórcio Knockout Gene Oklahoma. Os autores agradecem Dan Stefanovic e Tanner Wheeler por sua valiosa ajuda durante a execução do projeto.

<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>

Não há conflitos de interesse declarados.

Neste estudo nós desenvolvemos um protocolo de utilização fácil, amigável que pode ser usado para perfis gene determinada expressão subjacente vários fenômenos biológicos. Neste protocolo, o RNA total foi isolado pela primeira vez e tratados com DNase I. Nosso método de isolamento de RNA foi drasticamente simplificada omitindo extrações fenol e utilizam resinas afinidade para limpar 5,6. Resumidamente, C. membranas elegans cutícula e células estavam rachados aberto por nematóides congelamento com nitrogênio líquido e moagem com resina molecular. Em seguida, o RNA extraído foi tratado com DNase I antes da síntese de cDNA. A etapa posterior foi adicionado para minimizar hibridizações inespecíficas; amostras de RNA contaminados com DNA genômico poderia introduzir interferências e produzem muitos falsos positivos. Em seguida, digite, selvagem e vsm-1 (ok1468) cDNAs mutantes foram marcados com Cy3 e Cy5 seqüências de captura e hibridizado em C. microarrays elegans obtidas através do programa GCAT. Este sistema é mais sensível do que produtos similares, e seu design de duas cores diminui o número de matrizes necessários, resultando em maior tempo e eficiência de custos 7,8. Além disso, este sistema não requer o equipamento especializado de outros sistemas similares, portanto, este procedimento poderia ser realizado em qualquer configuração padrão de laboratório 7. Terceiro, imagens fluorescentes série hibridizadas foram analisados ​​utilizando o software open source MAGICTool, e perfis de expressão gênica foram criados. MAGICTool é um programa fácil de usar que pode ser facilmente utilizado por indivíduos de todos os níveis de experiência, a partir da graduação ao pós-doutorado. No entanto, o desempenho ideal requer disponibilidade de memória de grande porte. Por último, genes candidatos obtidos pela análise microarray foram validados com PCR em tempo real. Embora a abordagem genômica é um método eficiente para o estudo de fenômenos biológicos, existem algumas limitações deve-se levar em consideração. Primeiro, apesar da especificidade deste protocolo de microarray, transcrições dentro de uma família são indistinguíveis uma da outra se o oligonucleotídeo impresso é retirado de sequências conservadas. PCR em tempo real compensa semelhanças dentro de uma família de genes e pode até mesmo distinguir entre isoformas específicas do mesmo gene. No entanto, com PCR em tempo real é um desafio para encontrar controles verdade que são expressos igualmente em todo tempo de vida de um organismo. Devido a isso, os resultados serão relativos. Uma abordagem proteômica é uma alternativa para a nossa técnica. Proteínas individuais podem ser isoladas por meio de eletroforese em gel 2D e identificado com espectrometria de massa. Nossos estudos mostram que especificamente muitos membros da família de proteínas do esperma Major (MSP) são induzidos em vsm-1 nematóides mutantes com maior densidade sináptica. MSPs são exclusivas para C. elegans e são responsáveis ​​pela maturação do oócito ea ovulação. Chai 1 tem demonstrado que a regulação do número e tamanho bouton pré-sináptica na junção neuromuscular em moscas de frutas é provável controlado por moléculas contendo domínios principal proteína do esperma. Estudos realizados por Tsuda e seus colegas demonstraram dois domínios principal proteína do esperma pode servir como ligantes para receptores Ephrine, desencadeando cascatas de sinalização do receptor tirosina quinase. Além disso, análise em tempo real PCR validados e reduzida resultados microarray para um membro da família msp, msp-32. Tomados em conjunto, o trabalho aqui apresentado representa uma análise do genoma de um dilema neurociência central, bem como o poder de iniciação científica no esforço científico.

<table border="1"><tbody><tr><td> Componente </td><td> Catalogo </td><td> Companhia </td></tr><tr><td> Mais tarde RNA </td><td> AM7020 </td><td> Ambion </td></tr><tr><td> Resina Grinding Molecular </td><td> 786-138 </td><td> Gbiosciences </td></tr><tr><td> RNeasy Mini Kit </td><td> 74104 </td><td> Qiagen </td></tr><tr><td> RNase Agarose LE </td><td> AM9040 </td><td> Ambion </td></tr><tr><td> NorthernMax Gly 10X Gel buffer Prep / Execução </td><td> 8678 </td><td> Ambion </td></tr><tr><td> RNA Millennium marcador </td><td> AM7150 </td><td> Ambion </td></tr><tr><td> Glioxal Amostra Loading Dye </td><td> 8551 </td><td> Ambion </td></tr><tr><td> DNase set </td><td> 79254 </td><td> Qiagen </td></tr><tr><td> RNeasy MinElute Kit </td><td> 74204 </td><td> Qiagen </td></tr><tr><td> RT Enzima e Tampão de reacção de 5X </td><td> RT300320 </td><td> Genisphere </td></tr><tr><td> Matriz 350 </td><td> W300180 </td><td> Genisphere </td></tr><tr><td> QIAquick Kit de Purificação PCR </td><td> 28104 </td><td> Qiagen </td></tr><tr><td> 20X SSC </td><td> 82021-4846 </td><td> VWR </td></tr><tr><td> DNA de esperma de salmão cortado </td><td> AM9680 </td><td> Ambion </td></tr><tr><td> SDS Solution </td><td> V6551 </td><td> Promega </td></tr><tr><td> TDT EM </td><td> 3860 </td><td> VWR </td></tr><tr><td> iScript Kit Síntese de cDNA </td><td> 170-8890 </td><td> BioRad </td></tr><tr><td> iQ SYBR Green 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

Sinapses são compostos de uma zona de pré-sináptica ativa na sinalização celular e um terminal pós-sináptico na célula-alvo. No caso de sinapses químicas, as mensagens são transportadas por neurotransmissores liberados a partir de terminais pré-sinápticos e recebidos por receptores nas células pós-sináptica. Nossa pesquisa anterior, em Caenorhabditis elegans tem mostrado que VSM-1 regula negativamente exocitose. Além disso, a análise das sinapses no VSM-1 mostraram que os animais mutantes faltando um completamente funcional conectividade VSM-1 aumentaram sináptica. Com base nestes resultados preliminares, que a hipótese de que C. elegans VSM-1 pode desempenhar um papel crucial na sinaptogênese. Para testar essa hipótese, duplo-rotulados análise microarray foi realizada, e os perfis de expressão gênica foram determinados. Primeiro, RNA total foi isolado, transcritas reversamente para cDNA e hibridizadas à microarrays de DNA. Então, em silico-análise de hibridização da sonda fluorescente revelou indução significativa de muitos genes que codificam membros da família maiores de esperma proteína (MSP) em mutantes com a sinaptogênese reforçada. MSPs são o principal componente do esperma em C. elegans e aparecem para sinalizar a maturação do oócito ea ovulação nematóide. Em moscas de frutas, Chai e seus colegas demonstraram que uma MSP-como moléculas regulam número bouton pré-sináptico e tamanho na junção neuromuscular. Além disso, a análise realizada por Tsuda e colegas de trabalho sugeriu que dois MSPs podem atuar como ligantes para os receptores e desencadear Ef receptor tirosina quinase cascatas de sinalização. Por último, análise em tempo real PCR confirmaram que o gene que codifica para a MSP-32 é induzida em vsm-1 (ok1468) mutantes. Tomados em conjunto, a pesquisa realizada por nosso laboratório demonstraram que vsm-1 mutantes têm um aumento significativo na densidade sináptica, o que poderia ser mediada por MSP-32 sinalização.

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

Análise de microarrays foi conduzido para determinar perfis de expressão genética em<em&gt; C. elegans</em&gt; E PCR em tempo real foi utilizada para validar e quantificar os dados microarray.

Determinar perfis de expressão genética em C. elegans Usando Microarray e PCR em tempo real

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

Research

JoVE Journal

Biology

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

22.9K visualizações.  Southwestern Oklahoma State University. Análise de microarrays foi conduzido para determinar perfis de expressão genética em C. elegans E PCR em tempo real foi utilizada para validar e quantificar os dados microarray.

Vídeo: 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.

Geração de transgênicos Estável C. elegans Usando microinjeção

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

Biologia

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.

Análise da expressão global de Gene usando uma plataforma Microarray Zebrafish Oligonucleotide

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.

Quantitativas Real-Time PCR usando o 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 ...

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.

Análise de Expressão Gênica em Emerald Borer Ash ( Agrilus planipennis) Usando Quantitativa em Tempo Real-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.

Perfil dos pré-micro RNAs e microRNAs usando Quantitative PCR em tempo real (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 ...

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.

Bioquímica e Alto Rendimento Avaliação microscópica de massa gorda em<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.

Anticorpo de coloração em<em&gt; C. Elegans</em&gt; Usando o &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 ...

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.

Linear Amplificação Mediada por PCR - Localização de elementos genéticos e Caracterização de Unknown Flanquear DNA

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.

Baseado-Probe-PCR em tempo real Abordagens para a medição quantitativa de 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 ...

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.

Análise qPCRTag - A High Throughput, PCR em Tempo Real Ensaio para Sc2.0 Genotipagem

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 ...