1. A Thermo Scientific Solaris qPCR Algorithm Design de Ensaio <ol><li> O algoritmo usado no projeto de Solaris qPCR sonda / primer pares ajusta o Tm de cada componente permitindo assim condições de ciclismo universal. Incorporação do MGB, ou menor ligação groove, metade na sonda aumenta o Tm e permite sondas mais curto para ser projetado para uma maior flexibilidade de ensaio e performance. Como seu nome sugere, MGBs são uma classe de antibióticos que podem se encaixar no sulco menor da dupla hélice do DNA. MGBs são anexados ao final 3 ou 5 de uma sonda de DNA. Em solução, a fluorescência repórter FAM da sonda se apaga por Eclipse Refrescante Dark. Quando a sonda se liga a uma seqüência alvo, a mudança resultante em conformação a sonda s 3-D permite que um forte sinal fluorescente. Ligação da sonda de DNA para seqüência alvo é estabilizado pelo MGB, que permite o uso de altamente específicas, as sondas mais curtos, mantendo a temperatura de fusão apropriado para PCR. Assim, a detecção do sinal ocorre durante a fase de recozimento de amplificação do alvo. Durante a etapa de extensão, a sonda MGB desassocia ea fluorescência é novamente apagado. Porque a sonda desassocia, ela não afeta a eficiência da PCR. </li><li> Outra característica do algoritmo Solaris é o uso de Superbases. Superbases são modificados bases que têm maior capacidade de ligação para melhorar a estabilidade da AT títulos e eliminar GG auto-associação em guanina seqüências ricas. Além disso, eles não saciar fluoróforos adjacentes conectado no terminal 5. Estas bases são seletivamente colocada, de acordo com o algoritmo, para expandir a disponibilidade de seqüências-alvo. Isto torna possível a utilização de seqüências, tais como as regiões GC ricos, que poderiam ser evitados na elaboração de primers e sondas. </li><li> Sempre que possível, o algoritmo Solaris incorpora regiões junção exon abrangendo no site de destino. Isso aumenta a especificidade, evitando a amplificação do DNA genômico contaminante. </li><li> Importante, o algoritmo também identifica uma seqüência consenso para todas as variantes conhecidas de emenda, de modo que todos estão cobertos por uma única sonda / set primer. Assim, apenas um ensaio é necessário para um determinado gene de interesse. </li><li> Finalmente, o Solaris qPCR algoritmo projeto Ensaio utiliza parâmetros muito rigorosos para considerações de design críticas, incluindo o conteúdo GC, comprimento, Tm, e trechos de nucleotídeos homogênea. As seqüências resultantes são analisados ​​para a especificidade BLAST usando três diferentes bases de dados: genômica, transcrição e pseudogene. </li></ol> 2. Obtenção Solaris Reagentes <ol><li> Para obter o apropriado Solaris reagentes, visite <a href="http://dharmacon.com">www.thermo.com / solaris</a> </li><li> Uma vez no site clique em &quot;Pesquisar Produto GENEius&quot;. Para ir diretamente para a busca de produtos GENEius, vá para <a href="http://www.dharmacon.com/catalogsearch/consolidatedsearch.aspx">www.thermo.com / SolarisSearch</a> </li><li> Clique na caixa de pesquisa de pesquisa e escolha do organismo de destino apropriado. </li><li> A pesquisa de produtos GENEius pode processar muitos identificadores gene diferente. Aqui, entrar em um dos termos de pesquisa sugeriram para encontrar o gene de interesse, incluindo Ref Seq de Adesão, a identificação do gene, o Symbol Gene, uma descrição do Gene, o ID Sanger ou número de Adesão, ou um número de catálogo. Em seguida, clique em &quot;Pesquisar&quot;. </li><li> Identificar o gene de interesse e clique em &quot;Select&quot; no lado esquerdo. Isto irá abrir uma janela que mostra os diferentes produtos Thermo Scientific disponíveis para o gene de interesse. Em &quot;Produtos&quot; selecione a opção &quot;Detecção de qPCR&quot; Tab. </li><li> Sob o título &quot;Ensaio Expressão Gênica Solaris&quot; título, você vai ver um bem concebido Solaris sonda / ensaio primer. Este ensaio vai cobrir uma emenda todas as variantes conhecidas de seu alvo gene. Escolha a quantidade desejada. Eles estão disponíveis em 100, 200, 400 e 1000 X 25 mL reações (se o ensaio é identificado como made-to-order, o tamanho do pacote de 100 não estará disponível). Clique em &quot;Add to Cart&quot;. </li><li> Em seguida, selecione o master mix apropriado para o termociclador que será usado. Para determinar qual master mix vai funcionar melhor com a máquina disponível, clique em &quot;Saiba quais Mix Master funciona melhor com o seu Cycler PCR!&quot; </li><li> Encontre o cycler qPCR que será usado no menu para determinar quais mix de usar. </li><li> Retornar à janela de busca de produtos GENEius, e selecione o master mix apropriado. O master mix vem em 100, 200, 400 e 1000 tamanhos reação. Clique em &quot;Add to Cart&quot;. </li><li> Genes de referência propostos e kits verso cDNA síntese também pode ser encomendado para uma análise completa qRT-PCR. </li><li> Quando os reagentes Solaris qPCR chegam, armazená-los a -20 ° C até que esteja pronto para uso. Os seguintes reagentes devem ser recebidos: Uma solução de 20X, contendo dois primers fornecidos a 800 nM cada e uma sonda gene específico previsto em 200 nm, sequinformações de referência para ambos os sonda e os primers e qPCR Gene Solaris Mix Master Expressão em uma concentração 2X. Solaris qPCR Ensaios Expressão Gênica são estáveis ​​por um período mínimo de 12 meses. Evite congelar repetido descongelamento. </li></ol> 3. qPCR configuração <ol><li> Iniciar o protocolo de qPCR com cDNA. RNA total deve ser extraído usando um método de coluna de spin. A Thermo Scientific Verso kit síntese de cDNA é recomendado para transcrição reversa para gerar cDNA. </li><li> Prepare-se para qPCR por descongelar os primers, sondas e master mix no gelo. Também tem em mãos PCR água grau e opaco placas qPCR branco, o que pode melhorar a sensibilidade do PCR em tempo real. </li><li> A mistura mestre contém todos os componentes para PCR em tempo real, incluindo: 1) DNA polimerase Thermo-Start, uma versão hot-início da DNA polimerase Thermoprime. Este tipo de polimerase é utilizada para prevenir não-específico de amplificação durante a reação, configurar e requer etapa de ativação de 95 ° C min 15. 2) dNTPs na mistura Mestre Solaris, dTTP substitui dUTP para maximizar a eficiência de amplificação 3) tampão de reação Proprietário, otimizado para trabalhar com Solaris primer / sonda ensaios. Este tampão de reação contém corante azul para aumentar o contraste entre reagente e plástico. 4) ROX, se o seu instrumento qPCR requer ROX. </li><li> Uma vez que as soluções têm descongelado, misture sacudindo o tubo (não vortex). Spin-los para baixo para evitar a perda de reagente. </li><li> Em seguida, preparar um tubo de 15 mL Falcon estéril para configurar o mix de reação para o gene de interesse, mais um para o gene de referência, ampliação, dependendo do número de amostras. Prepare o suficiente tal que três repetições podem ser preparados para cada amostra, incluindo um modelo de controle não (NTC) e as amostras de curva padrão, se o método de curva padrão será usado para análise. </li><li> Para cada amostra rodar em uma placa de 96 poços, misture o seguinte: 5 mL da qPCR Solaris 2X Mix Master, 0,5 mL do conjunto Solaris Primer / Probe (20X), e PCR água grau tal que o volume final de reação, uma vez o cDNA é adicionado será de 25 mL. Para cada amostra rodar em uma placa de 384 poços misturar os seguintes: 12,5 mL da qPCR Solaris 2X Mix Master, 1,25 mL do conjunto Solaris Primer / Probe (20X), e PCR água grau tal que o volume final de reação, uma vez que o cDNA é adicionado será de 10 mL. </li><li> Usando um pipetador multicanal, transferir a mistura de mestre para nos poços apropriados da placa. O corante azul incluídos no master mix vai ajudar no progresso de pipetagem de rastreamento nas placas brancas. Em seguida, adicionar o template 1-5 mL cDNA (para uma placa de 96 poços) ou 1-2 mL de modelo de cDNA (para uma placa de 384 poços). Pipeta cima e para baixo para misturar. Coloque a placa em uma centrífuga e spin para baixo para remover as bolhas, pois estas irão interferir com as leituras de fluorescência. </li><li> Programa do termociclador: A enzima deve ser ativado com um 95 ° C 15 min (1 ciclo). Em seguida, 40 ciclos de 95 ° C, a desnaturação por 15 segundos, e recozimento e extensão a 60 ° C por 60 segundos. </li><li> Coloque a placa no instrumento qPCR. Iniciar o programa. </li></ol> Resultados representativos qPCR usando Solaris <img src="/files/ftp_upload/1700/1700fig1.jpg" alt="Figura 1" /> Figura 1. Ensaios Solaris dar a detecção confiável, mesmo em concentrações muito baixas de entrada, a julgar pela eficiência da PCR e valores r2. 10 dez diluições de cDNA sintetizado a partir seqüência sintética amplicon RNA ou DNA seqüência amplicon foi amplificado em um instrumento 7900HT ABI usando Solaris qPCR Assay Expressão Gênica para F2RL1 ou CDC20, respectivamente. As curvas de log escala de amplificação e curvas de nível são mostrados juntamente com o desempenho de cada ensaio, incluindo a eficiência, o valor de r2, faixa dinâmica de 10 log10 diluições, eo limite mínimo de detecção. <img src="/files/ftp_upload/1700/1700fig2.jpg" alt="Figura 2" /> Figura 2. Assays Solaris dar resultados consistentes até mesmo entre diferentes pesquisadores e laboratórios. siRNAs segmentação ALDOA e PPIB, bem como um controle não-alvo (NTC), foram transfectados em células HeLa em 100, 10, 1 e 0,1 nM concentrações final. As células foram colhidas e RNA total isolado 48 horas pós-tratamento. cDNA foi sintetizado usando Thermo Scientific Verso Kit Síntese de cDNA. Uma alíquota do cDNA foi amplificado em dois laboratórios separados geograficamente usando Solaris qPCR Ensaios Expressão Gênica para a detecção de ALDOA, PPIB, e GAPDH em um LightCycler Roche 480 (384 poços) da plataforma. Knockdown foi calculado usando o método ΔΔCq (normalizados para GAPDH referência gene e NTC tratados com células). Os mesmos níveis de knockdown foram demonstradas para alvos tanto gene entre os dois pesquisadores em ambos os locais.

Os autores deste artigo são funcionários da Thermo Scientific.

Thermo Scientific Ensaios Solaris qPCR fornecer um método simples e melhor para a realização de análise de qPCR. Usando um algoritmo de alta severidade, incorporando tecnologia e MGB Superbases, esses conjuntos de primers e sonda maximizar a fidelidade ea robustez dos experimentos qPCR. Uma cartilha e definir sonda reconhece todas as variantes conhecidas emenda de um determinado gene. O site de destino, sempre que possível, inclui as regiões exon abrangendo, para garantir que o DNA genômico não é amplificado. Estas características tornam possível executar várias amostras simultaneamente, reduzindo a quantidade de tempo necessário para realizar experimentos complexos. Desde a mistura principal é azul, é possível usar placas brancas opacas, que melhoram a sensibilidade do qPCR, sem perder de vista a pipetagem. Tomados em conjunto, as características do projeto do Ensaio qPCR Solaris fazer este ensaio qPCR romance um ensaio simples, mas de alto desempenho que é específica e robusta.

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

O Solaris qPCR Assay expressão do gene é um novo tipo de primer / sonda conjunto, projetado para simplificar o processo de qPCR, mantendo a sensibilidade e precisão do ensaio. Estes conjuntos primer / sonda são pré-concebidos para&gt; 98% dos genomas humano e do rato e melhorias característica importante das tecnologias anteriormente disponíveis. Estas melhorias foram possíveis em virtude de um algoritmo de design inovador, desenvolvido por especialistas Thermo Scientific bioinformática. Várias características convenientes foram incorporados Ensaio qPCR Solaris para agilizar o processo de realizar quantitativa PCR em tempo real. Primeiro, o protocolo é semelhante ao de alternativas comumente empregados, por isso os métodos utilizados durante a qPCR são susceptíveis de ser familiar. Segundo, a mistura principal é azul, o que torna a configuração das reações qPCR mais fácil de rastrear. Em terceiro lugar, as condições de ciclos térmicos são os mesmos para todos os ensaios (genes), tornando possível para executar muitas amostras em um tempo e reduzindo o potencial de erro. Finalmente, a sonda e informações de seqüência de primer são fornecidos, simplificando o processo de publicação. Aqui, nós demonstrar como obter os reagentes apropriados Solaris usando o recurso de busca GENEius produto encontrado no site da ordenação (www.thermo.com / solaris) e como utilizar os reagentes para a realização de qPCR Solaris usando o método de curva padrão.

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

O Solaris qPCR Ensaios Expressão Gênica são novos pré-concebidas qPCR primer / sonda combinações projetado para simplificar o processo de qPCR sem sacrificar a especificidade e robustez do ensaio.

Quantitativas Real-Time PCR usando o Thermo Scientific Solaris qPCR Assay

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

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Vídeo: 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.

Microvolume determinação da concentração de proteína utilizando o NanoDrop 2000c Espectrofotômetro

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

Biologia

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

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

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

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

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

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

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

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

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

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

Perfil dos pré-micro RNAs e microRNAs usando Quantitative PCR em tempo real (qPCR) Arrays

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

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 quantificação Microvolume de Ácidos Nucléicos

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.

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

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

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.

Genotipagem de amostras de plantas e animais, sem purificação DNA Antes

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.

Quantitativa, análise em tempo real da Base de Dados de atividade de reparo de excisão em lisados ​​celulares Utilizando Lesão específicos Beacons Molecular

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.

Medições em tempo real de proteína de membrana: Receptor Interações Usando Superfície 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 ...

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

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

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

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

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

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

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

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

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

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...