1. El Thermo Scientific Solaris ensayo qPCR diseño de algoritmos <ol><li> El algoritmo utilizado en el diseño de Solaris qPCR sonda / primer pares se ajusta a la Tm de cada componente lo que permite condiciones universales ciclismo. Obligatoria la incorporación de la MGB, o surco menor, fracción en la investigación aumenta el Tm y permite una más corta que las sondas diseñadas para la flexibilidad y el rendimiento del ensayo mayor. Como su nombre indica, MGBs son una clase de antibióticos que pueden encajar en el surco menor de la hélice del ADN. MGBs se adjunta al final de 3 a 5 de una sonda de ADN. En la solución, la fluorescencia reportero FAM de la sonda se apaga por saciar la Eclipse oscuro. Cuando la sonda se une a una secuencia objetivo, el cambio resultante en 3-D la conformación de la sonda s permite que una señal fluorescente fuerte. La unión de la sonda de ADN de secuencia objetivo es estabilizado por el MGB, que permite el uso de muy específicas, las sondas más cortas, manteniendo la temperatura de fusión adecuado para PCR. Por lo tanto, la detección de la señal se produce durante la fase de recocido de amplificación de la diana. Durante la etapa de extensión, la sonda MGB se disocia y la fluorescencia se apaga de nuevo. Debido a que la sonda se disocia, no afecta la eficiencia de la PCR. </li><li> Otra característica del algoritmo de Solaris es el uso de superbases. Superbases se modifican las bases que han mejorado la capacidad de unión para mejorar la estabilidad de los bonos A y eliminar GG libre asociación en guanina secuencias ricas. Por otra parte, no apagar fluoróforos adyacentes conectados a la terminal 5. Estas bases son colocados de forma selectiva, de acuerdo con el algoritmo, para ampliar la disponibilidad de la secuencia blanco. Esto hace posible el uso de secuencias, como las regiones ricas en GC, que de otra forma se evitaría en el diseño de cebadores y sondas. </li><li> Siempre que sea posible, el algoritmo de Solaris incluye las regiones de unión exón de expansión en el sitio de destino. Esto aumenta la especificidad al evitar la amplificación de ADN genómico contaminante. </li><li> Es importante destacar que el algoritmo también identifica una secuencia consenso de todas las variantes de empalme conocidos, de tal manera que todos están cubiertos por una sola sonda / primer set. Por lo tanto, sólo un ensayo que se necesita para un determinado gen de interés. </li><li> Por último, el sistema operativo Solaris ensayo qPCR algoritmo de diseño utiliza parámetros muy estrictos para las consideraciones de diseño críticos, entre ellos el contenido de GC, la duración, Tm, y tramos de nucleótidos homogénea. Las secuencias resultantes se analizaron para BLAST especificidad con tres diferentes bases de datos: genómica, el expediente académico, y pseudogen. </li></ol> 2. La obtención de reactivos Solaris <ol><li> Para obtener los reactivos de Solaris, visite <a href="http://dharmacon.com">www.thermo.com / solaris</a> </li><li> Una vez en el sitio web, haga clic en &quot;Búsqueda de Productos GENEius&quot;. Para ir directamente a la búsqueda de productos GENEius, vaya a <a href="http://www.dharmacon.com/catalogsearch/consolidatedsearch.aspx">www.thermo.com / SolarisSearch</a> </li><li> Haga clic en la caja de consulta de búsqueda y elegir el organismo de destino apropiado. </li><li> La búsqueda de productos GENEius puede procesar muchos identificadores de genes diferentes. Aquí se pueden introducir uno de los términos de búsqueda sugeridos para encontrar el gen de interés, incluyendo Ref. Sec. de adhesión, la identificación de genes, el símbolo del gen, una descripción de genes, la identificación o el número de Sanger, o un número de catálogo. A continuación, haga clic en &quot;Buscar&quot;. </li><li> Identificar el gen de interés y haga clic en &quot;Seleccionar&quot; en el lado izquierdo. Esto abrirá una ventana que muestra los diferentes productos de Thermo Scientific para el gen de interés. En &quot;Productos&quot; seleccione la opción &quot;Detección de qPCR&quot; Tab. </li><li> En el marco del &quot;Ensayo de Solaris expresión génica&quot; la partida, podrás ver un óptimo diseño de la sonda Solaris / ensayo manual. Este ensayo un cubrirá todas las variantes conocidas de empalme de genes objetivo. Elija la cantidad deseada. Están disponibles en 100, 200, 400 y 1000 X 25 reacciones l (si el ensayo se identifica como hecho a la medida, el tamaño del paquete de 100 no estará disponible). Haga clic en &quot;Añadir al carro&quot;. </li><li> A continuación, seleccionar la combinación adecuada para dominar el termociclador que se utilizará. Para determinar qué mezcla maestra que funcionará mejor con la máquina disponible, haga clic en &quot;Find a Mix Master, que funciona mejor con su termociclador de PCR!&quot; </li><li> Buscar la cicladora qPCR que se utilizará en el menú para determinar cuál es la combinación de su uso. </li><li> Volver a la ventana de búsqueda GENEius producto, y seleccionar la combinación maestro adecuado. La mezcla maestra viene en 100, 200, 400 y 1000 tamaños de reacción. Haga clic en &quot;Añadir al carro&quot;. </li><li> Genes de referencia sugerido y los kits de verso cDNA síntesis también se puede pedir para una completa QRT-PCR análisis. </li><li> Cuando los reactivos Solaris qPCR llegar, almacenarlos a -20 ° C hasta que esté listo para su uso. Los siguientes reactivos deben ser recibidos: Una solución de 20X, con dos primers siempre a 800 nM cada uno y una sonda del gen específico establecido en 200 nm, secuenciadorescia de información tanto para la sonda y cebadores, y Solaris Gene qPCR Expresión Master Mix en una concentración de 2X. Solaris qPCR ensayos de expresión de genes son estables durante un mínimo de 12 meses. Evite congelar y descongelar repetidas. </li></ol> 3. qPCR configuración <ol><li> Comience el protocolo qPCR con cDNA. ARN total se extrajo utilizando un método spin columna. El Thermo Scientific kit Verso cDNA síntesis se recomienda para la transcripción inversa para generar ADNc. </li><li> Prepárese para qPCR por el deshielo de los cebadores, las sondas, y mezcla maestra sobre el hielo. También tener a mano agua grado PCR y qPCR opaco placas blancas, que pueden mejorar la sensibilidad de la PCR en tiempo real. </li><li> La mezcla maestra contiene todos los componentes para PCR en tiempo real, incluyendo: 1) la ADN polimerasa Thermo-Start, una versión de arranque en caliente de la ADN polimerasa Thermoprime. Este tipo de polimerasa se utiliza para evitar la amplificación no específica durante la reacción de instalar y requiere la activación de paso a 95 ° C 15 min. 2) en la mezcla de dNTPs Solaris Maestro, dTTP reemplaza dUTP para maximizar la eficiencia de amplificación 3) tampón de reacción de propiedad, optimizado para trabajar con Solaris cartilla / sonda de ensayos. Este tampón de reacción contiene un tinte azul para aumentar el contraste entre el reactivo y el plástico. 4) ROX, si su instrumento qPCR requiere ROX. </li><li> Una vez que las soluciones se han descongelado, mezcle agitando el tubo (no vortex). Girar hacia abajo para evitar la pérdida de reactivo. </li><li> Luego, prepare un tubo de 15 ml estériles Falcon para establecer la mezcla de reacción para el gen de interés, más uno para el gen de referencia, la ampliación en función del número de muestras. Prepare suficiente como para que tres réplicas se pueden preparar para cada muestra, incluyendo un control sin plantilla (NTC) y las muestras de la curva estándar, si el método de la curva estándar se utiliza para el análisis. </li><li> Para cada muestra se ejecutan en una placa y 96, mezclar los siguientes: 5 l de la mezcla de Solaris qPCR 2X Master, 0,5 l de la versión de Solaris Primer / sonda conjunto (20 veces), y el agua PCR grado tal que el volumen final de reacción, una vez el ADNc se agrega será de 25 mL. Para cada muestra se ejecutan en una placa de 384 y mezclar los siguientes: 12,5 l de la mezcla de Solaris qPCR 2X Master, 1,25 l de la versión de Solaris Primer / sonda conjunto (20 veces), y el agua como PCR grado que el volumen final de reacción, una vez que el cDNA se agrega será de 10 mL. </li><li> Utilizando una pipeta multicanal, la transferencia de la mezcla maestra a los pocillos correspondientes de la placa. El tinte azul incluido en la mezcla maestra ayudará en el progreso de pipeteo de seguimiento en las placas blancas. A continuación, agregue el 5.1 l plantilla de cDNA (de una placa y 96) o 2.1 L de cDNA plantilla (para una placa de 384 pocillos). Pipeta de arriba a abajo para mezclar. Colocar la placa en una centrífuga y centrifugar para eliminar las burbujas, ya que estos interfieren con las lecturas de fluorescencia. </li><li> Programa del termociclador: La enzima se debe activar con un 95 º C 15 minutos (1 ciclo). Luego 40 ciclos de 95 ° C, la desnaturalización de 15 segundos, y el recocido y extensión a 60 ° C durante 60 segundos. </li><li> Colocar la placa en el instrumento qPCR. Iniciar el programa. </li></ol> Representante qPCR resultados utilizando Solaris <img src="/files/ftp_upload/1700/1700fig1.jpg" alt="Figura 1" /> Figura 1. Ensayos de Solaris dar una detección fiable incluso en concentraciones muy bajas de entrada, a juzgar por la eficiencia de la PCR y los valores de r2. Diez de 10 diluciones de cDNA sintetizado a partir de la secuencia sintética de amplificación del ARN o la secuencia de amplificación de ADN se amplificó en un instrumento ABI 7900HT con Solaris Gene qPCR ensayo de expresión de F2RL1 o Cdc20, respectivamente. Las curvas de amplificación escala logarítmica, y las curvas de calibración se muestran junto con el rendimiento de cada ensayo en particular la eficiencia, el valor de r2, el rango dinámico de 10 log10 diluciones y el límite inferior de detección. <img src="/files/ftp_upload/1700/1700fig2.jpg" alt="Figura 2" /> Figura 2. Ensayos de Solaris dar resultados consistentes, incluso entre los diferentes investigadores y laboratorios. siRNAs dirigidos ALDOA y PPIB, así como un control no-objetivo (NTC), fueron transfectadas en células HeLa a 100 nm, 10, 1 y 0,1 la concentración final. Las células fueron cosechadas y el ARN total aislado 48 horas después del tratamiento. cDNA fue sintetizado utilizando Thermo Scientific Kit Verso cDNA Síntesis. Una alícuota del cDNA se amplificó en dos laboratorios separados geográficamente, utilizando Solaris qPCR ensayos de expresión genética para la detección de ALDOA, PPIB y GAPDH en un LightCycler de Roche 480 (de 384 pozos) de la plataforma. Caída se calculó utilizando el método ΔΔCq (normalizado a GAPDH genes de referencia y las células tratadas con NTC). Los mismos niveles de caída se manifestó en favor tanto de los objetivos de genes entre los dos investigadores en ambos sitios.

Los autores de este artículo son empleados de Thermo Scientific.

Thermo Scientific Solaris ensayos qPCR un método sencillo, mejor para la realización de análisis qPCR. Utilizando un algoritmo de alto rigor, la incorporación de la tecnología y la MGB superbases, estos conjuntos de cebadores y sonda de maximizar la fidelidad y la solidez de los experimentos de qPCR. Un primer y sonda conjunto reconoce todas las variantes conocidas de empalme de un determinado gen. El sitio de destino, siempre que sea posible, incluye las regiones que abarca el exón, para asegurarse de que el ADN genómico no se amplifica. Estas características hacen posible el funcionamiento de numerosas muestras de forma simultánea, lo que reduce la cantidad de tiempo necesario para realizar experimentos complejos. Dado que la mezcla principal es azul, es posible el uso de placas de color blanco opaco, lo que mejora la sensibilidad de la PCR cuantitativa, sin perder de vista la pipeta. En conjunto, las características de diseño del ensayo qPCR Solaris hacen de este ensayo qPCR novela un ensayo simple, pero de alto rendimiento que es específica y sólida.

quantitative real-time pcr using the thermo scientific solaris qpcr assay

El Solaris Gene qPCR ensayo de expresión es un nuevo tipo de juego de cebadores / sonda, diseñada para simplificar el proceso de qPCR mientras se mantiene la sensibilidad y la precisión de la prueba. Estos conjuntos de cebadores / sonda se pre-diseñados para&gt; 98% de los genomas humano y del ratón y mejoras de las características importantes de las tecnologías ya disponibles. Estas mejoras han sido posibles gracias a un algoritmo de diseño novedoso, desarrollado por expertos en bioinformática Thermo Scientific. Varias funciones convenientes han sido incorporados en el ensayo qPCR Solaris para agilizar el proceso de llevar a cabo cuantitativas PCR en tiempo real. En primer lugar, el protocolo es similar a otras alternativas comúnmente empleados, por lo que los métodos utilizados en qPCR es probable que esté familiarizado. En segundo lugar, la mezcla maestra es de color azul, lo que hace que la configuración de las reacciones qPCR más fácil de seguir. En tercer lugar, las condiciones de ciclos térmicos son los mismos para todos los ensayos (los genes), lo que permite ejecutar muchas muestras a la vez y reducir las posibilidades de error. Finalmente, la sonda y la información de la secuencia de los cebadores se proporcionan, lo que simplifica el proceso de publicación. En este sentido, se muestra cómo obtener la reactivos de Solaris con la función de búsqueda de productos GENEius encontrar en el sitio web de pedidos (www.thermo.com / solaris) y el uso de los reactivos para la realización de Solaris qPCR utilizando el método de la curva estándar.

Watch this Scientific Journal Video about Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay at JoVE.com

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

El Solaris qPCR ensayos de expresión génica son nuevas pre-diseñados qPCR primer / sonda combinaciones diseñadas para simplificar el proceso de qPCR sin sacrificar la especificidad y la robustez de la prueba.

Cuantitativas PCR en tiempo real utilizando el Thermo Scientific Solaris qPCR ensayo

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

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49.6K vistas.  Thermo Scientific Solaris qPCR Products.  日本語 

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

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

Concentración de proteínas microvolumen Determinación mediante el NanoDrop 2000c Espectrofotómetro

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

Investigación

Biología

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

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

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

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

Análisis de la expresión génica en el barrenador esmeralda del fresno ( Agrilus planipennis) Utilizando Real-PCR cuantitativa en tiempo

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

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

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

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

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

Perfiles de Pre-micro ARN y microRNAs utilizando cuantitativa en tiempo real PCR (qPCR) Arrays

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

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop microvolumen cuantificación de ácidos nucleicos

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

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

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

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

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

La determinación de perfiles genéticos de expresión en C. elegans Utilización de microarrays y PCR en tiempo real

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

El genotipado de muestras de plantas y animales, sin previa purificación del ADN

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

Cuantitativo, análisis en tiempo real de la actividad de reparación por escisión de base en lisados ​​celulares que utilizan la lesión específicos Molecular beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

Mediciones en tiempo real de proteínas de membrana: Receptor interacciones utilizando resonancia de plasmones superficiales (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

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

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

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

PCR en tiempo real basada en sondeos Enfoques para la medición cuantitativa de microRNAs

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

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

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

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

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 ...