Os autores gostariam de agradecer A Viksø-Nielsen (Novozymes) para o dom das enzimas celulolíticas. Este trabalho foi financiado pelo FP7 RENOVAÇÃO e por projetos BBSRC BB/G016178 e BB/G016194.

1. Preparação das amostras Nós normalmente trabalham com hastes de qualquer herbáceas ou plantas lenhosas. No caso de materiais de herbáceas que crescem as plantas até o vencimento, e após o desenvolvimento da semente que coletamos hastes seco uma vez a senescência está completa. As hastes são cortadas em quatro segmentos mm e colocadas em 2 frascos ml com três bolas de moagem. No caso de materiais lenhosos, as amostras são moídas para serragem grossa de madeira com um arquivo do curso e, em seguida, colocada em um moinho de bolas para processamento posterior. As amostras são colocadas em um rack dentro da moagem / formatação estação (Figura 1). Esta estação seqüencialmente grinds, mixagens, e pesa cada amostra. O número de repetições necessárias por amostra é estabelecida pelo teste do material moído em uma série de experimentos preliminares em que a variabilidade intrínseca para um tamanho de partícula certo é determinada. Os 2 mL são perfurados na parte inferior e os materiais são dispensados ​​pela vibração do frasco sobre o bem selecionado. O braço oscilante é controlado por um feedback de equilíbrio que permite a distribuição exata do pó. O robô dispensa 4 mg / poço em uma análise standard e 4 repetições são necessários na maioria dos casos. Isto permite que 20 amostras de plantas / placa a ser analisado. 2. Pré-tratamento Depois que as amostras são formatados, as placas de 96 poços são processadas por um sistema automatizado líquido de manejo (Figura 2). Aqui, um pré-tratamento é realizado em leve os materiais vegetais, adicionando 350 mL de ácido ou soluções alcalinas e aquecimento da placa em um bloco adaptado. Para evitar evaporação durante a análise, os 96 pratos são bem selado com um mate silicone. A temperatura eo tempo de pré-tratamento pode ser modificado de acordo com os materiais em estudo. A temperatura máxima, no entanto, é de 100 ° C. Um procedimento alternativo para o teste pré-tratamentos mais duras é para pré-tratamento dos materiais fora de linha e eliminar o pretreatmet do processo. 3. Hidrólise <ol><li> Após os materiais são pré-tratados, o robô automaticamente remove a solução de pré-tratamento e lavagens, todos os poços, 5 vezes com 850 mL tampão acetato 25 mM. Os enxaguatórios são realizadas pela adição de tampão à solução de pré-tratamento e, posteriormente, removendo 50% do volume total depois de permitir que os sólidos na amostra para resolver. A altura da aspiração é fixado em metade da massa de líquido para evitar a aspiração de sólidos do fundo do poço, não há perda desprezível de sólidos usando essa abordagem. Após estas lavagens do pH no poço é de 4,5 eo material está pronto para a hidrólise enzimática. </li><li> Uma mistura de enzimas contendo uma solução de celulases e hemicelulases é adicionado pelo robô. Em cada poço, 850 mL da mistura de enzimas é dispensada ea placa é transferida para uma 50 ° C agitando incubadora. A hidrólise padrão é realizada por 8 h, mas desta vez pode ser adaptado para a finalidade do experimento. O carregamento da enzima padrão usado para gramíneas é de 7 FPU / g de material. </li></ol> 4. Detecção de açúcares redutores Determinação de açúcares redutores liberados após a hidrólise foi realizada utilizando uma versão modificada do 3-metil-2-benzothiazolinone zona hídrica método (MTBH) 3. MBTH foi selecionado como o método mais adequado, porque era mais fácil de automatizar e menos suscetível a interferência de tais compostos como proteínas. Nós modificamos este método altamente sensível para uso na plataforma robótica para que pudesse quantificar com precisão os açúcares nas concentrações presentes no hidrolisado de biomassa, com um volume final de 250 mL que é adequado para um padrão de 96 placa óptica bem. Todos os passos a seguir são executadas automaticamente e três determinações independentes dos açúcares redutores foram realizadas para cada reação sacarificação. <ol><li> 30 mL de hidrolisado foi retirado da placa de poço profundo e misturado com 45 mL de tampão acetato 25 mM de sódio em um contornou PCR placa de 96 poços. </li><li> 25 mL de NaOH 1N e 50 ul de uma solução contendo 0,43 mg / ml MBTH e 0,14 mg / ml DTT foram adicionados. </li><li> Após a mistura, a placa de PCR foi aquecida a 60 ° C por 20 min em um termociclador ou dispositivo similar que oferece exatamente a mesma quantidade de calor para todos os 96 poços. </li><li> A reação resultante foi transferido para uma placa óptica. </li><li> Adicionar 100 ul do reagente oxidante (0,2% FeNH4 (SO4) 2, 0,2% de ácido sulfâmico e 0,1% HCl). Misture bem e deixe a desenvolver por pelo menos 1 h. </li><li> Cada chapa deve conter também as reações padrão de 50 nmol, 100 e 150 nmol de glicose nmol. As placas ópticas são lidas a 620 nm. </li></ol> 5. Resultados representativos: Exemplos de diferentes tipos de análises são apresentados na figura 3 e 4. Todos os resultados foram obtidos utilizando a plataforma automatizada. Figura 3 reapresenta o aumento de açúcares redutores liberados por 8 h de incubação poplar terreno com quantidades crescentes de enzimas celulolíticas. A Figura 4 apresenta os efeitos do ácido e pré-tratamento alcalino em amostras de álamo. O pré-tratamento NaOH é mais eficiente que o pré-tratamento H2SO4 diluído em facilitar a liberação de açúcares durante a hidrólise. A quantidade de açúcares medidos após hidrólise diminui quando o pré-tratamento é realizado com concentrações superiores a NaOH 1M. <img src="/files/ftp_upload/3240/3240fig1.jpg" alt="Figura 1" /> Figura 1. Estação Robotic para moer e 96 de formatação bem de biomassa <img src="/files/ftp_upload/3240/3240fig2.jpg" alt="Figura 2" /> Figura 2. Estação de tratamento de líquido para a análise de Alto Rendimento sacarificação <img src="/files/ftp_upload/3240/3240fig3.jpg" alt="Figura 3" /> Figura 3. Efeito de cargas enzima diferente sobre a libertação de equivalentes redutores de açúcar a partir de amostras de álamo <img src="/files/ftp_upload/3240/3240fig4.jpg" alt="Figura 4" /> Figura 4. Açúcares Efeito de diferentes pré-tratamentos na sacarificação de amostras de álamo. A. liberado após pretreament com diferentes porcentagens de H2SO4 a 90 ° C por 30 minutos. Açúcares B. liberado após pré-tratamentos NaOH à mesma temperatura e tempo do que o pré-tratamento ácido .

<ol>
	<li>Carpita, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Biogenesis+of+the+Cell+Walls+of+Grasses.">Structure and Biogenesis of the Cell Walls of Grasses.</a> Annu Rev Plant Physiol Plant Mol Biol. 47, 445-476 (1996).</li><li>Gomez, L. D., Steele-King, C. G., McQueen-Mason, 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=Sustainable+liquid+biofuels+from+biomass:+the+writing's+on+the+walls.">Sustainable liquid biofuels from biomass: the writing's on the walls.</a> New Phytol. 178, 473-485 (2008).</li><li>Anthon, G. E., Barrett, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+reducing+sugars+with+3-methyl-2-benzothiazolinonehydrazone.">Determination of reducing sugars with 3-methyl-2-benzothiazolinonehydrazone.</a> Anal Biochem. 305, 287-289 (2002).</li><li>Gomez, L. D., Whitehead, C., Barakate, A., Halpin, C., McQueen-Mason, 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=Automated+saccharification+assay+for+determination+of+digestibility+in+plant+materials.">Automated saccharification assay for determination of digestibility in plant materials.</a> Biotechnol Biofuels. 3, 23-23 (2010).</li></ol>

Variations of the standard saccharification protocol can be used in the same platform for determining the activity of cellulolytic enzymes (i.e. by variation of the enzyme concentrations used in a plate as well as using paper as substrate); comparison of the efficiency of several enzyme mixtures on a specific material; time course for saccharification; etc.
A standard saccharification protocol to compare the saccharification potential in different plant materials involves an eight hour hydrolysis (Figures 3 and 4). Most of the plant materials analysed requires four replicates. Under these conditions, the platform can process 80 samples/day. This analysis is being used to screen large populations of barley, maize, and brachypodium in order to establish variability in saccharification potential and the genes involved in its determination4.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> Moagem e pesagem robô </td><td> Labman Automação </td><td></td></tr><tr><td> 2 ml micro tubo com tampas </td><td> Sarstedt Ltd </td><td> 72,694 </td></tr><tr><td> 5 milímetros de aço inoxidável contas </td><td> Qiagen Ltd </td><td> 69989 </td></tr><tr><td> 1,2 ml Abgene quadrados bem pratos de armazenamento </td><td> Fisher Scientific Ltd </td><td> TUL-050-050C </td></tr><tr><td> Whatman tampa esteira por 96 quadrados placas bem </td><td> Fisher Scientific Ltd </td><td> 7704-0104 </td></tr><tr><td> Robot hanling líquido </td><td> Tecan Group Ltd. </td><td> Liberdade Evo 200 </td></tr><tr><td> O ácido sulfúrico </td><td> Fisher Scientific Ltd </td><td> S/9231/PB17 </td></tr><tr><td> Hidróxido de sódio </td><td> Fisher Scientific Ltd </td><td> BPE359-500 </td></tr><tr><td> Acetato de sódio </td><td> Sigma-Aldrich </td><td> S8750-500G </td></tr><tr><td> Ácido acético </td><td> Fisher Scientific Ltd </td><td> A/0420/PB17 </td></tr><tr><td> Novozyme 188 </td><td> Novozyme </td><td> DCN00214 </td></tr><tr><td> Celluclast 1.5L </td><td> Novozyme </td><td> CCN03122 </td></tr><tr><td> 96 bem cheia placas PCR saia </td><td> Sarstedt Ltd </td><td> 72.1980.202 </td></tr><tr><td> D-Glicose </td><td> Fisher Scientific Ltd </td><td> G/0450/53 </td></tr><tr><td> 3-Metil-2-hidrato de cloridrato de hidrazona benzothiazolinone </td><td> Sigma-Aldrich </td><td> 129739-25G </td></tr><tr><td> DL-ditiotreitol </td><td> Sigma-Aldrich </td><td> D9163-1G </td></tr><tr><td> Corning microplaca com 96 fundo bem plana </td><td> Fisher Scientific Ltd </td><td> TKT-521-050H </td></tr><tr><td> De amônio sulfato de ferro dodeca (III) </td><td> Sigma-Aldrich </td><td> F1668-250G </td></tr><tr><td> O ácido sulfâmico </td><td> Sigma-Aldrich </td><td> 242772-500G </td></tr><tr><td> Ácido clorídrico </td><td> Fisher Scientific Ltd </td><td> 12462-0026 </td></tr></tbody></table>

high-throughput saccharification assay for lignocellulosic materials

Polissacarídeos que compõem a biomassa vegetal lignocelulósica pode ser dividido para produzir uma variedade de açúcares que posteriormente podem ser utilizados no estabelecimento de uma biorefinaria. Essas matérias-primas que constituem uma nova plataforma industrial, que seja sustentável e carbono neutro, para substituir a actual dependência de combustíveis fósseis. A recalcitrância à desconstrução observado em materiais lignocelulósicos é produzido por várias propriedades intrínsecas das paredes celulares das plantas. Celulose cristalina é incorporado na matriz de polissacarídeos, tais como xilanas e arabinoxilanos, e toda a estrutura é envolto pela fenólicos polímero da lignina, que também é difícil de digerir 1. A fim de melhorar a digestibilidade dos materiais vegetais precisamos descobrir os principais gargalos para a sacarificação das paredes das células e também mutante tela e populações reprodutoras para avaliar a variabilidade na sacarificação 2. Estas tarefas requerem uma abordagem de alto rendimento e aqui apresentamos uma plataforma analítica que pode realizar a análise sacarificação em um formato placa de 96 poços. Esta plataforma foi desenvolvida para permitir o rastreamento de digestibilidade da lignocelulose de grandes populações de espécies de plantas variadas. Nós temos reduzido os volumes de reação para pré-tratamento gentil, hidrólise enzimática parcial de açúcar e determinação, para permitir que um grande número de ser avaliado rapidamente em um sistema automatizado. Esta plataforma automatizada trabalha com quantidades de miligramas de biomassa, realizando moagem, sob condições controladas para reduzir os materiais vegetais a uma granulometria padronizada de forma reproduzível. Depois que as amostras são moídas, o robô automatizado dispensa a formatação quantidades especificadas e registradas de material nos poços correspondentes da placa de 96 poços profundos (Figura 1). Normalmente, nós dispensar o mesmo material em 4 poços de ter 4 repetições para análise. Uma vez que as placas são preenchidos com o material vegetal no layout desejado, eles são movidos manualmente para uma estação de manipulação de líquidos (Figura 2). Nesta estação as amostras são submetidas a um pré-tratamento leve com um ácido diluído ou alcalina e incubadas a temperaturas de até 90 ° C. A solução de pré-tratamento é posteriormente removida e as amostras são lavadas com tampão para devolvê-los a um pH adequado para a hidrólise. As amostras são então incubados com uma mistura de enzimas por um período de tempo variável, a 50 ° C. Uma alíquota é retirada do hidrolisado e os açúcares redutores são automaticamente determinados pelo método colorimétrico MBTH.

Watch this Scientific Journal Video about High-throughput Saccharification Assay for Lignocellulosic Materials at JoVE.com

High-throughput Saccharification Assay for Lignocellulosic Materials

Um método simples e rápido para determinar o potencial de sacarificação de um grande número de amostras de biomassa vegetal é descrito. A plataforma automatizada para esta análise envolve a preparação da biomassa de plantas para análise em 96 placas bem e do desempenho posterior de hidrólise pré-tratamento e quantificação dos açúcares liberados.

Alto rendimento Assay Sacarificação para materiais lignocelulósicos

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

high-throughput-saccharification-assay-for-lignocellulosic-materials

Research

JoVE Journal

Biology

13.2K visualizações.  University of York. Um método simples e rápido para determinar o potencial de sacarificação de um grande número de amostras de biomassa vegetal é descrito. A plataforma automatizada para esta análise envolve a preparação da biomassa de plantas para análise em 96 placas bem e do desempenho posterior de hidrólise pré-tratamento e quantificação dos açúcares liberados.

Vídeo: High-throughput Saccharification Assay for Lignocellulosic Materials

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

Alto rendimento Yeast Tela superexpressão Plasmid

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Biologia

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

RNA Secundária Previsão Estrutura Usando SHAPE de alta capacidade

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

De alta capacidade de Triagem funcional usando um caseiro Dual-brilho ensaio de luciferase

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Um Ensaio de Ligação de Alto Rendimento II do MHC para análise quantitativa de Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

De alta capacidade de Titulação de luciferase expressando recombinantes Vírus

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recuperação de Zebrafish Adulto Corações para aplicações de alto rendimento

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

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 ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Detecção de alvos miRNA em métodos de alta capacidade utilizando o ensaio 3&#39;LIFE

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

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 Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

High Throughput<em&gt; Danio rerio</em&gt; Energia Despesas Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...