Los autores desean agradecer a un Viksø-Nielsen (Novozymes) por el don de las enzimas celulolíticas. Este trabajo fue financiado por el 7PM RENOVACIÓN y por los proyectos de BBSRC BB/G016178 y BB/G016194.

1. Preparación de las muestras Normalmente trabajo con tallos herbáceos o de cualquiera de las plantas leñosas. En el caso de las materias herbáceas que crecen las plantas a la madurez, y después de desarrollo de la semilla que obtenemos los tallos secos, una vez se haya completado la senescencia. Los tallos se cortan en segmentos de 4 mm y se colocan en viales de 2 ml con tres bolas de molienda. En el caso de materiales leñosos, las muestras se muelen a grueso aserrín de madera con un archivo de curso y se coloca en un molino de bolas para su posterior procesamiento. Las muestras se colocan en un estante en la molienda / formato de la estación (Figura 1). Esta estación forma secuencial muele, se mezcla, y un peso de cada muestra. El número de repeticiones necesarias para cada muestra se estableció mediante pruebas de los materiales del suelo en una serie de experimentos preliminares en donde se determina la variabilidad intrínseca de un tamaño de partícula determinado. Los viales de 2 ml se perforan en la parte inferior y los materiales son distribuidos por la vibración de la ampolla en el pozo seleccionado. El brazo de vibración es controlada por una retroalimentación del equilibrio que permite la dosificación precisa de la pólvora. El robot dispensa 4 mg / pocillo en un análisis estándar y 4 repeticiones son necesarias en la mayoría de los casos. Esto permite que 20 muestras de plantas / placa para ser analizados. 2. Pretratamiento Una vez que las muestras tienen el formato, las placas de 96 pozos son procesadas por un sistema automatizado de manejo de líquidos (Figura 2). Aquí un pretratamiento suave se realiza en los materiales vegetales mediante la adición de 350 l de soluciones ácidas o alcalinas y el calentamiento de la placa en un edificio adaptado. Para evitar la evaporación durante el análisis, el 96 y placas se sellan con un mate de silicona. La temperatura y el tiempo de la pre-tratamiento se puede modificar de acuerdo a los materiales objeto de estudio. La temperatura máxima, sin embargo, es de 100 ° C. Un procedimiento alternativo para la prueba más dura pretratamientos es dar tratamiento previo a los materiales fuera de línea y eliminar la pretreatmet del proceso. 3. Hidrólisis <ol><li> Después de que los materiales son tratados previamente, el robot elimina automáticamente la solución pre-tratamiento, y se lava, todos los pozos, 5 veces con 850 l de tampón acetato 25 mM. Los enjuagues se llevan a cabo mediante la adición de tampón de la solución pre-tratamiento, y posteriormente retirar el 50% del volumen total después de permitir que los sólidos en la muestra a un acuerdo. La altura de la aspiración es igual a la mitad de la masa líquida para evitar la aspiración de sólidos desde el fondo del pozo, hay una pérdida insignificante de los sólidos utilizando este enfoque. Después de estas soluciones el pH en el pozo es de 4,5 y el material está listo para la hidrólisis enzimática. </li><li> Una mezcla de enzimas que contienen una solución de celulasas y hemicelulasas se añade por el robot. En cada pozo, 850 l de mezcla de enzimas se dispensa y la placa se mueve en un 50 ° C incubadora de agitación. La hidrólisis se lleva a cabo estándar de 8 horas, pero esta vez se puede adaptar a la finalidad del experimento. La carga de la enzima estándar utilizado para los pastos es de 7 FPU / g de material. </li></ol> 4. La detección de azúcares reductores Determinación de azúcares reductores liberados después de la hidrólisis se realizó mediante una modificación de 3-metil-2-benzotiazolinona hidrozona (MTBH) el método 3. MBTH fue seleccionado como el método más adecuado, porque era el más fácil de automatizar y el menos susceptible a la interferencia de estos compuestos como las proteínas. Hemos modificado este método altamente sensible para su uso en la plataforma robótica para que pueda cuantificar con precisión los azúcares en las concentraciones presentes en los hidrolizados de biomasa, con un volumen final de 250 l, que es adecuado para un nivel óptico de 96 pocillos. Todos los pasos se realizan automáticamente y tres determinaciones independientes de los azúcares reductores se llevaron a cabo para cada reacción de sacarificación. <ol><li> 30 l de hidrolizado fue tomada de la placa de pozo profundo y se mezcla con 45 l de 25 mM de tampón acetato de sodio en una falda PCR 96 pocillos. </li><li> 25 l de NaOH 1N y 50 l de una solución que contiene 0,43 mg / ml y 0,14 MBTH TDT mg / mL. </li><li> Después de mezclar, la placa de PCR se calentó a 60 ° C durante 20 minutos en un termociclador o un dispositivo similar que ofrece exactamente la misma cantidad de calor para los 96 pozos. </li><li> La reacción resultante se transfirió a una placa óptica. </li><li> Añadir 100 ml de reactivo oxidante (0,2% FeNH4 (SO4) 2, 0,2% de ácido sulfámico y el 0,1% HCl). Mezclar bien y dejar a desarrollar por lo menos durante 1 h. </li><li> Cada plato debe contener también las reacciones estándar de 50 nmol, 100 y 150 nmol glucosa nmol. Las placas se leen óptica a 620 nm. </li></ol> 5. Los resultados representativos: Ejemplos de diferentes tipos de análisis se presentan en la figura 3 y 4. Todos los resultados se obtuvieron mediante el uso de la plataforma automatizada. Figura 3 representa el aumento de los azúcares reductores liberados por 8 h incubaciones de álamo suelo con cantidades crecientes de enzimas celulolíticas. La Figura 4 muestra los efectos del ácido alcalino y pretratamiento de las muestras de álamo. El pretratamiento con NaOH es más eficiente que el pretratamiento con H2SO4 diluido para facilitar la liberación de los azúcares en la hidrólisis. La cantidad de azúcares medidos después de la hidrólisis disminuye cuando el tratamiento previo se realiza con concentraciones superiores a 1M NaOH. <img src="/files/ftp_upload/3240/3240fig1.jpg" alt="Figura 1" /> Figura 1. Robótico de la Estación para la molienda y el 96 y el formato de la biomasa <img src="/files/ftp_upload/3240/3240fig2.jpg" alt="Figura 2" /> Figura 2. Estación de manejo de líquidos para el análisis de rendimiento de sacarificación de alta <img src="/files/ftp_upload/3240/3240fig3.jpg" alt="Figura 3" /> Figura 3. Efecto de las cargas de enzimas diferentes en el lanzamiento de la reducción equivalente de azúcar a partir de muestras de álamo <img src="/files/ftp_upload/3240/3240fig4.jpg" alt="Figura 4" /> Figura 4. Efecto de pretratamientos diferentes en la sacarificación de las muestras de álamo. Azúcares A. en libertad después de pretreament con diferentes porcentajes de H2SO4 a 90 ° C durante 30 minutos. B. Azúcares en libertad después de pretratamientos de NaOH a la misma temperatura y el tiempo que el pretratamiento con 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> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> Molienda y pesaje robot </td><td> Labman Automatización </td><td></td></tr><tr><td> 2 ml micro tubo con tapas </td><td> Sarstedt Ltd </td><td> 72.694 </td></tr><tr><td> 5 cuentas de acero inoxidable de </td><td> Qiagen Ltd </td><td> 69989 </td></tr><tr><td> 1,2 ml ABgene cuadrados y las placas de almacenamiento </td><td> Fisher Scientific Ltd </td><td> TUL-050-050C </td></tr><tr><td> Whatman tapa alfombra para 96 ​​placas cuadradas </td><td> Fisher Scientific Ltd </td><td> 7704-0104 </td></tr><tr><td> Robot hanling líquido </td><td> Tecan Group Ltd. </td><td> Libertad Evo 200 </td></tr><tr><td> De ácido sulfúrico </td><td> Fisher Scientific Ltd </td><td> S/9231/PB17 </td></tr><tr><td> De hidróxido de sodio </td><td> Fisher Scientific Ltd </td><td> BPE359-500 </td></tr><tr><td> Acetato de sodio </td><td> Sigma-Aldrich </td><td> S8750-500G </td></tr><tr><td> El 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 y placas de PCR falda </td><td> Sarstedt Ltd </td><td> 72.1980.202 </td></tr><tr><td> D-glucosa </td><td> Fisher Scientific Ltd </td><td> G/0450/53 </td></tr><tr><td> 3-metil-2-benzotiazolinona hidrazona hidratos de clorhidrato de </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 de 96 inferior y plana </td><td> Fisher Scientific Ltd </td><td> TKT-521-050H </td></tr><tr><td> Amonio y hierro (III) sulfato dodecahidrato </td><td> Sigma-Aldrich </td><td> F1668-250G </td></tr><tr><td> Sulfámico ácido </td><td> Sigma-Aldrich </td><td> 242 772-500g </td></tr><tr><td> Ácido clorhídrico </td><td> Fisher Scientific Ltd </td><td> 12462-0026 </td></tr></tbody></table>

high-throughput saccharification assay for lignocellulosic materials

Polisacáridos que forman la biomasa lignocelulósica planta se puede dividir para producir una amplia gama de azúcares que puedan ser utilizados posteriormente en el establecimiento de una biorrefinería. Estas materias primas que constituyen una nueva plataforma industrial, que sea sostenible y neutral de carbono, para reemplazar a la actual dependencia de combustibles fósiles. La obstinación de la deconstrucción observado en los materiales lignocelulósicos es producido por varias propiedades intrínsecas de las paredes celulares vegetales. Celulosa cristalina está integrada en los polisacáridos de la matriz como xilanos y arabinoxilanos, y toda la estructura está recubierta por el fenólicos polímero de la lignina, que es también difícil de digerir 1. Con el fin de mejorar la digestibilidad de las materias vegetales que tenemos que descubrir los principales obstáculos para la sacarificación de las paredes celulares y mutante de la pantalla y las poblaciones de mejoramiento para evaluar la variabilidad en la sacarificación 2. Estas tareas requieren un enfoque de alto rendimiento y aquí se presenta una plataforma de análisis que puede realizar un análisis de sacarificación en un formato de placa de 96 pocillos. Esta plataforma ha sido desarrollada para permitir la detección de la digestibilidad de la materia leñosa de las grandes poblaciones de especies de plantas variadas. Hemos reducido los volúmenes de reacción para el pretratamiento suave, la hidrólisis enzimática parcial y la determinación de azúcar, para permitir que un gran número para ser evaluados rápidamente en un sistema automatizado. Esta plataforma automatizada trabaja con cantidades de miligramos de biomasa, rendimiento de molienda por bolas en condiciones controladas para reducir el material vegetal a un tamaño de partícula estándar de una manera reproducible. Una vez que las muestras son de tierra, el robot automatizado formato dispensa cantidades específicas y del material registrado en los pocillos correspondientes de la placa de 96 pozos profundos (Figura 1). Normalmente, dispensar el mismo material en 4 pozos de tener 4 repeticiones para el análisis. Una vez que las placas se llena con el material vegetal en el diseño deseado, que se mueven manualmente a una estación de manejo de líquidos (Figura 2). En esta estación las muestras son sometidas a un tratamiento previo leve, ya sea con ácido diluido o alcalino y se incubaron a temperaturas de hasta 90 ° C. La solución de pre-tratamiento se retira posteriormente y las muestras se lavan con tampón para volver a un pH adecuado para la hidrólisis. Las muestras se incubaron con una mezcla de enzimas durante un período variable de tiempo a 50 ° C. Una alícuota se toma a partir del hidrolizado y los azúcares reductores se determina automáticamente por el 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

Un método simple, rápido para determinar el potencial de la sacarificación de un gran número de muestras de biomasa vegetal se describe. La plataforma automatizada para este análisis consiste en la preparación de la biomasa vegetal para su análisis en placas de 96 pozos y la realización posterior de pretratamiento, hidrólisis y la cuantificación de los azúcares liberados.

De alto rendimiento de sacarificación de ensayo para materiales 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 vistas.  University of York. Un método simple, rápido para determinar el potencial de la sacarificación de un gran número de muestras de biomasa vegetal se describe. La plataforma automatizada para este análisis consiste en la preparación de la biomasa vegetal para su análisis en placas de 96 pozos y la realización posterior de pretratamiento, hidrólisis y la cuantificación de los azúcares liberados.

Video: High-throughput Saccharification Assay for Lignocellulosic Materials

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

De alto rendimiento de cristalización de proteínas de membrana Utilizando el Método lipídico Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Investigación

Biología

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.

ARN de predicción de estructura secundaria Utilizando SHAPE de alto rendimiento

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.

Selección de alto rendimiento funcional utilizando un doble resplandor de ensayo de luciferasa Homemade

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.

Un ensayo de unión de alto rendimiento MHC II para el análisis cuantitativo del péptido epítopos

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 Alto Rendimiento La titulación de luciferasa que expresan virus recombinantes

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.

Recuperación de adultos de pez cebra Corazones para aplicaciones de alto rendimiento

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.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

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.

La detección de los genes miARN objetivos en de alto rendimiento utilizando el ensayo 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.

Análisis qPCRTag - Un alto rendimiento, Tiempo real ensayo PCR para Sc2.0 Genotipado

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; Ensayo gasto energético

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