Este trabalho foi financiado por uma bolsa da National Science Foundation (conceder número MCB-0447750).

<ol>
	<li>Clark, K. A., Krysan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol:+An+improved+high-throughput+method+for+generating+tissue+samples+in+96-well+format+for+plant+genotyping+(Ice-Cap+2.0).">Protocol: An improved high-throughput method for generating tissue samples in 96-well format for plant genotyping (Ice-Cap 2.0).</a> Plant Methods. 3, 8-8 (2007).</li><li>Krysan, P. I. c. e. -. c. a. p. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+capturing+plant+tissue+samples+for+genotype+analysis.">A high-throughput method for capturing plant tissue samples for genotype analysis.</a> Plant Physiol. 135, 1162-1169 (2004).</li><li>Jander, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+map-based+cloning+in+the+post-genome+era.">Arabidopsis map-based cloning in the post-genome era.</a> Plant Physiol. 129, 440-450 (2002).</li><li>Liljegren, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHATTERPROOF+MADS-box+genes+control+seed+dispersal+in+Arabidopsis.">SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis.</a> Nature. 404, 766-770 (2000).</li><li>Buechel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+A-type+ARABIDOPSIS+RESPONSE+REGULATORS+in+meristem+maintenance+and+regeneration.">Role of A-type ARABIDOPSIS RESPONSE REGULATORS in meristem maintenance and regeneration.</a> Eur. J. Cell. Biol. 89, 279-284 (2010).</li><li>Krysan, P. J., Jester, P. J., Gottwald, J. R., Sussman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Arabidopsis+mitogen-activated+protein+kinase+kinase+kinase+gene+family+encodes+essential+positive+regulators+of+cytokinesis.">An Arabidopsis mitogen-activated protein kinase kinase kinase gene family encodes essential positive regulators of cytokinesis.</a> Plant Cell. 14, 1109-1120 (2002).</li><li>Bush, S. M., Krysan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iTILLING:+a+personalized+approach+to+the+identification+of+induced+mutations+in+Arabidopsis.">iTILLING: a personalized approach to the identification of induced mutations in Arabidopsis.</a> Plant Physiol. 154, 25-35 (2010).</li><li>Comai, L., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING:+practical+single-nucleotide+mutation+discovery.">TILLING: practical single-nucleotide mutation discovery.</a> Plant J. 45, 684-694 (2006).</li><li>Henikoff, S., Till, B. J., Comai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TILLING.+Traditional+mutagenesis+meets+functional+genomics.">TILLING. Traditional mutagenesis meets functional genomics.</a> Plant Physiol. 135, 630-636 (2004).</li><li>McCallum, C. M., Comai, L., Greene, E. A., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+induced+local+lesions+IN+genomes+(TILLING)+for+plant+functional+genomics.">Targeting induced local lesions IN genomes (TILLING) for plant functional genomics.</a> Plant Physiol. 123, 439-442 (2000).</li></ol>

Não há conflitos de interesse declarados.

O método Ice Cap-apresentada aqui permite que um cientista para coletar amostras de tecido de centenas ou mesmo milhares, de plantas individuais em um único dia e eficiente extrair o DNA genômico das amostras para uso em reações de genotipagem. Dando um cientista individual a capacidade de genótipo milhares de mudas em um curto período de tempo, o método Ice Cap-tem o potencial para facilitar uma série de experiências genéticas que não seriam práticos para executar. Vários exemplos são fornecidos abaixo. <ol><li> Mapeamento genético. Numa escala de mapeamento genético pode ser acelerada se o pesquisador é capaz de identificar eventos de recombinação dentro de um intervalo específico ao longo do cromossomo 3. Se este intervalo envolve uma distância muito curta genética, então os eventos de recombinação será relativamente raros na população de mapeamento segregação. Por esta razão, pode ser necessário para milhares de tela de plantas individuais para encontrar a desejada reindivíduos combinant. Este processo pode ser bastante acelerada com a utilização de Ice Cap-. Mesmo na era do genoma re-seqüenciamento, é ainda necessário demonstrar diretamente que uma mutação é diretamente responsável por um fenótipo de interesse. Nestes casos, será necessário segregar as mutações de interesse longe de outras mutações intimamente ligada a fim de provar uma mutação candidato é realmente causador. Nestas situações, Ice Cap-se proporcionar um método eficiente para a quebra de ligação entre um par de mutações intimamente ligados. </li><li> Multi-mutantes. Tornou-se comum para pesquisadores que estudam Arabidopsis para combinar alelos mutantes de vários loci distintos em um único indivíduo, a fim de superar a redundância funcional 4-6. Como exemplo, considere um caso em que se deseja combinar alelos mutantes de quatro loci diferentes em uma única planta por meio de cruzamento genético. Para este projeto, será necessário para gerar umn indivíduo que é heterozigoto para estas quatro loci como parte do esquema de cruzamento. Quando isso quadruple-heterozigoto semente sets, o homozigoto quadruple é esperado para estar presente a uma taxa de um em cada 256 na população resultante da progênie. Triagem para essa pessoa rara pelos métodos tradicionais seria muito trabalhoso. Por comparação, usando Ice Cap-para este projeto proporcionaria o cientista com uma estratégia eficiente para identificar o homozigoto rara quadruplicar em uma única geração. </li><li> iTILLING. Nosso laboratório descreveu recentemente uma nova abordagem para screening para a presença de mutações induzidas em genes de interesse em 7 Arabidopsis. Este método é uma variação de uma técnica estabelecida chamado Tilling que é usado para localizar mutações em populações ordenada de indivíduos tratados com um agente mutagénico química 8-10. Nós chamamos nosso método iTILLING porque é uma versão individualizada de cultivo que permite que um indcientista ividual para realizar algo que tradicionalmente tem exigido uma equipe de pesquisa grande para ser concluído. Ice Cap-faz parte integrante do método iTILLING 7, conforme descrito abaixo. </li></ol> A idéia por trás iTILLING é produzir uma população mutante efêmera, que contrasta com as populações duráveis ​​mutante criado para projetos de Tilling tradicionais. Para iTILLING, sementes de uma população bulked M2 são semeadas em 5-10 blocos Ice Cap, e DNA é extraído utilizando o método de Ice Cap-. Arabidopsis mudas são então colocadas a 4 ° C, enquanto triagem de mutação é realizada utilizando o DNA Ice Cap- preps. Mudas de tomate são armazenadas a 12 ° C.. Uma vez que mudas de portadores de mutações desejadas tenham sido identificadas, elas são recuperadas das placas Ice Cap-e transferidos para o solo. iTILLING não se destina a fornecer uma população mutante de longa duração para uma comunidade de pesquisa toda a tela. Em vez disso, iTILLING fornece uma estratégia pela qualum cientista pode tela de uma população mutante personalizado para mutações em um punhado de genes com rapidez, eficiência e com um custo relativamente baixo. Foram encontradas Ice Cap-de ser um método eficaz para o cultivo e genotipagem Arabidopsis, tomate e arroz. Esperamos que outras espécies de plantas devem também ser passíveis de Ice Cap-. Uma limitação na variedade de espécies que podem ser acomodados por Ice-Cap é o tamanho das sementes. Apenas as plantas com sementes pequenas o suficiente para caber em poços de uma placa de 96 poços será compatível com Ice Cap-na sua configuração actual.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comments (opcional) </td></tr><tr><td> GenoGrinder </td><td> Spex / CertiPrep, Metuchen, NJ </td><td> 2000 Geno / Grinder </td><td> Máquina para agitar Placas Root para a extração de DNA </td></tr><tr><td> ABgene Thermo-Sealer </td><td> ABgene EUA </td><td> AB-0384 </td><td> Manual da máquina de vedação térmico utilizado para Placas Root antes de rompimento do tecido </td></tr><tr><td> Centrífuga equipado com portadores de microplacas balançando </td><td> Beckman Coulter </td><td> Allegra 25R </td><td> Qualquer centrífuga com as operadoras de microplacas balançando seria adequado </td></tr><tr><td> Micropartículas Dispenser de microplacas </td><td> Biotek www.biotek.com </td><td> AF1000A </td><td> Dispensador de líquido automatizado para microplacas enchimento </td></tr><tr><td> Bialetti marca17x11 polegadas assadeira comerciais de grande porte </td><td> Alvo Department Store </td><td> www.bialetti.com </td><td> Esta folha de cookie específico tem a profundidade ideal para Ice Cap- </td></tr><tr><td> Custom Made rack de metal </td><td> Loja de hardware </td><td></td><td> Veja o texto para descrição completa </td></tr><tr><td> 33 caixa de armazenamento Litro de plástico </td><td> Alvo Department Store </td><td> Sterilite Marca </td><td> Para Ice Cap-Fountain. Caixa de armazenamento de plástico grande o suficiente para segurar a assadeira 17x11 cm </td></tr><tr><td> 30 litros por hora Bomba Fountain </td><td> Sunterra </td><td> 104506 </td><td> Bomba submersível pequena fonte disponível em lojas da fonte do jardim </td></tr><tr><td> Mangueira para bomba da fonte </td><td> Qualquer empresa de fornecimento de laboratório </td><td></td><td> Mangueira compatível com a bomba da fonte </td></tr><tr><td> Braçadeira de plástico </td><td> Loja de hardware </td><td></td><td> Braçadeira para segurar mangueira para cookiesfolha </td></tr><tr><td> Limpar as tampas de plástico da Falcon fundo Microtest plana de 96 poços placas de poliestireno </td><td> BD Falcon www.bdbiosciences.com </td><td> 351172 </td><td> Limpar as tampas de plástico para cobrir Placas Ice Cap-Mudas </td></tr><tr><td> 3M esparadrapo micropore </td><td> Fisher Scientific </td><td> 19-027-761 </td><td></td></tr><tr><td> 3/32-inch diâmetro esferas de aço inoxidável </td><td> Hartford Technologies, Rocky Hill, CT </td><td> 034-006-1K </td><td> Usada para romper o tecido da raiz após a colheita </td></tr><tr><td> 3 polegadas elásticos de cabelo </td><td> Mercearia local </td><td></td><td> Usado para proteger Placa de raiz para Placa de Mudas </td></tr><tr><td> 4 milímetros de diâmetro espetos de madeira </td><td> Mercearia local </td><td></td><td> Estes espetinhos são tipicamente usados ​​para preparar espetinhos </td></tr><tr><td> Fisher Scientific Nunc marca de 1 ml placas de filtro sem fritas </td><td> Fisher Scientific </td><td> 278012 </td><td> Placas de 96 poços de Mudas com furos no fundo. </td></tr><tr><td> Fisherbrand 96-Bem Chimney Tall-Placa PCR </td><td> Fisher Scientific </td><td> 14-230-242 </td><td> Placas de 96 poços Root </td></tr><tr><td> Agar - cultura de células testados </td><td> Sigma-Aldrich </td><td> A1296 </td><td></td></tr><tr><td> 4 Morpholinoethanesulfonic ácido (MES) </td><td> Sigma-Aldrich </td><td> M2933 </td><td></td></tr><tr><td> Murashige e Skoog (MS) mistura de sal basal </td><td> Sigma-Aldrich </td><td> M-5524 </td><td></td></tr><tr><td> Calor Peel fácil vedação folha </td><td> ABgene EUA </td><td> AB-0745 </td><td> Filme calor de vedação para vedação placas Root antes de rompimento do tecido </td></tr><tr><td> Duck Tape Embalagem Marca Extra Wide, 3,00 &quot;x 54,6 m </td><td> OfficeMax www.officemax.com </td><td> Item no. 21242139 </td><td> Fita adesivavedação de microplacas contendo extratos de DNA para o armazenamento </td></tr><tr><td> Rolo de vedação </td><td> Bio-Rad </td><td> MSR-0001 </td><td> Mão ferramenta de rolamento para pressionar fita de vedação adesiva para microplacas </td></tr><tr><td> De 96 poços bloco de metal térmica </td><td> Cole-Parmer </td><td> 36400-66 </td><td> Usado para congelar as Placas de raiz para a colheita de tecidos </td></tr><tr><td> Papel de filtro Whatman </td><td> Whatman Internacional </td><td> 1002 090 </td><td> Usado para esterilização de sementes de superfície </td></tr><tr><td> Descartáveis ​​Pasteur Pipeta </td><td> Fisher Scientific </td><td> 13-678-6A </td><td> 5 ¾ &quot;descartáveis ​​pipeta Pasteur utilizado para a transferência de sementes para Placa de Mudas </td></tr><tr><td> Pyrex assadeira (20 cm x 30 cm) </td><td> Alvo Department Store </td><td></td><td> Usado para construir um banho de gelo / etanol seco. </td></tr></tbody></table>

ice-cap: a method for growing arabidopsis and tomato plants in 96-well plates for high-throughput genotyping

Está se tornando comum para os cientistas de plantas para desenvolver projetos que requerem a genotipagem de grande número de plantas. O primeiro passo em qualquer projeto de genotipagem é coletar uma amostra de tecido de cada planta individual. A abordagem tradicional para essa tarefa é para as plantas uma amostra-at-a-tempo. Se alguém deseja centenas genótipo ou milhares de indivíduos, no entanto, usando esta estratégia resulta em um gargalo significativo no pipeline genotipagem. O método Ice Cap-que descrevemos aqui oferece uma solução de alto rendimento a este desafio, permitindo que um cientista para coletar o tecido de vários milhares de mudas em um único dia, 1,2. Este nível de vazão é tornada possível pelo fato de que o tecido é colhido a partir de plantas 96-at-a-tempo, ao invés de um-em-um-tempo. O método Ice Cap-fornece uma plataforma integrada para a realização de um crescimento de mudas, a colheita de tecidos e extração de DNA. A base para Ice Cap-se o crescimento de seedlings em um par empilhadas de placas de 96 poços. Os poços da placa superior contêm fichas de ágar em que o crescimento de mudas individuais germinar. As raízes crescem para baixo através dos meios de ágar, sair da placa superior através de um buraco, e passar para uma água mais baixo da placa que contém. Para a colheita de tecidos para a extração de DNA, a água na parte inferior da placa contendo tecido raiz é rapidamente congelado, enquanto as mudas na placa superior permanecer em temperatura ambiente. A placa superior é, então, descoladas da placa inferior, resultando em um prato com 96 amostras de raízes tecido congelado em gelo e uma placa com 96 mudas viáveis. A técnica é chamada de &quot;Ice Cap-&quot; porque usa gelo para capturar o tecido radicular. A placa de 96 poços contendo as mudas pode, então, embrulhados em papel alumínio e transferidos para baixa temperatura. Este processo suspende um maior crescimento das mudas, mas não afeta a sua viabilidade. Pois a análise de genótipo foi concluída, as mudas com o genótipo desejado pode ser transferido do p de 96 poçostarde para o solo para a propagação ainda mais. Temos demonstrado a utilidade do método de Ice Cap-usando Arabidopsis thaliana, tomate, arroz e mudas. Esperamos que o método deve também ser aplicável a outras espécies de plantas com sementes pequenas o suficiente para caber nas cavidades da placas de 96 poços.

Watch this Scientific Journal Video about Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping at JoVE.com

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping | Biology | Video

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

O método Ice Cap-permite cultivar plantas em placas de 96 poços e não-destrutiva do tecido raiz colheita de cada muda. DNA extraído dessa raiz tecido pode ser usado para reações de genotipagem. Descobrimos que Ice Cap-funciona bem para<em&gt; Arabidopsis thaliana</em&gt;, Tomate, mudas e arroz.

Ice Cap: Um Método para Crescer Arabidopsis Plantas e tomate em placas de 96 poços para High-Throughput Genotipagem

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

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Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

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22.7K visualizações.  University of Wisconsin-Madison. O método Ice Cap-permite cultivar plantas em placas de 96 poços e não-destrutiva do tecido raiz colheita de cada muda. DNA extraído dessa raiz tecido pode ser usado para reações de genotipagem. Descobrimos que Ice Cap-funciona bem para Arabidopsis thaliana, Tomate, mudas e arroz.

Vídeo: Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pyrosequencing: Um método simples para genotipagem Accurate

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Biologia

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

Um método de alta taxa de transferência para Zebrafish criopreservação de espermatozóides e In Vitro Fertilização

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Um método rápido de alta capacidade para Ribonucleoproteínas Mapping (RNPs) no pré-mRNA Humanos

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Medição Caenorhabditis elegans Expectativa de Vida em 96 placas de microtitulação Bem

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

Um Método de alta capacidade para medição de Taxa de filtração glomerular em ratos conscientes

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Medindo espacial e temporal Ca<sup&gt; 2 +</sup&gt; Sinais em Arabidopsis Plantas

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Rastreio de alto rendimento para moduladores químicos de Genes Post-transcriptionally Regulamentados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Medição de resistência de Estresse Oxidativo<em&gt; Caenorhabditis elegans</em&gt; Em placas de 96 poços de microtitulação

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

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 ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

De alto rendimento CRISPR Vector construção e caracterização de DNA Modificações pela geração de raízes de tomateiro Cabeludo

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...