1。的Thermo Scientific的Solaris定量PCR检测设计算法<ol><li>在设计的Solaris qPCR的探针/引物对所使用的算法调整每个组件从而使通用循环条件的Tm。团的MGB探针，或轻微的凹槽约束力，在探头的基团增加以旧换新，并允许更短的设计更大的灵活性和性能检测的探头。顾名思义，MGBs是一类抗生素，可以容纳到DNA双螺旋的小沟。 MGBs连接到一个DNA探针的3或5月底。 FAM记者荧光探针在溶液中，是由Eclipse暗淬灭淬火。当探针结合到靶序列，在探头第3条的三维构象的变化，允许一个强烈的荧光信号。靶序列的DNA探针结合的稳定的MGB探针，它允许使用高度特异性的，更短的探针，而保持适当的熔融温度为PCR。因此，信号检测过程中发生的目标扩增的退火阶段。在延伸，MGB探针不赞成和荧光又是不灭的。因为探头不赞成，不影响PCR的效率。 </li><li>在Solaris算法的另一个特点是使用Superbases。 Superbases被修改，增强结合能力，以改善对AT债券的稳定和消除GG鸟嘌呤丰富的序列自我协会的基地。此外，他们不解渴5总站连接相邻的荧光团。这些基地选择性地，根据算法，扩大目标序列的可用性。这使得它可以使用，否则将避免在设计引物和探针的序列，如GC丰富的地区，。 </li><li>只要有可能，在Solaris算法采用外显子交界处，在目标网站跨越地区。这增加了避免污染物的基因组DNA扩增的特异性。 </li><li>重要的是，该算法还确定了所有已知的剪接变异体的共识序列，等，都是由一个单一的探针/引物覆盖。所以，只有一个实验是需要一个感兴趣的特定基因。 </li><li>最后，在Solaris的qPCR实验设计算法采用了非常严格的关键设计考虑的参数包括GC含量，长度，TM，和同质核苷酸的延伸。结果序列分析使用3种不同的数据库的特殊性的冲击：基因组，转录和假。 </li></ol> 2。获取的Solaris试剂<ol><li>要获得适当的Solaris试剂，请<a href="http://dharmacon.com">访问www.thermo.com / Solaris</a>的</li><li>一旦在网站上点击“GENEius产品搜索”。要直接去GENEius产品搜索，请访问<a href="http://www.dharmacon.com/catalogsearch/consolidatedsearch.aspx">www.thermo.com / SolarisSearch</a> </li><li>点击在搜索查询框，并选择适当的目标有机体。 </li><li> GENEius产品搜索，可以处理许多不同的基因标识。在这里，输入建议的搜索条件，寻找感兴趣的基因，其中包括加入参考序列，基因编号，基因符号，基因描述，桑格ID或加入号码，或目录号之一。然后，点击“搜索”。 </li><li>确定感兴趣的基因，并点击“选择”在左侧。这将打开一个窗口，显示不同的Thermo感兴趣的基因科技产品。根据“产品”，选择“定量PCR检测”选项卡。 </li><li>的“Solaris基因表达检测”的标题下，你会看到一个优化设计的Solaris探针/引物检测。此法将涵盖所有已知的基因目标的剪接变异体。选择所需的数量。他们在100，200，400， 1000 × 25μL的反应（100包大小，如果检测到，将无法使用）。点击“添加到购物车”。 </li><li>下一步，选择适当的热循环仪将用于主组合。要确定哪位高手组合将与现有的机器最好，单击“查找出最好与你的PCR扩增仪主混音作品！” </li><li>寻找定量PCR基因扩增仪，将在菜单上使用，以确定哪些组合使用。 </li><li>返回GENEius产品搜索窗口，并选择适当的主结构。主结构，100，200，400和1000的反应大小。点击“添加到购物车”。 </li><li>建议参考的基因和Verso cDNA合成试剂盒也可以订购一个完整的QRT - PCR分析。 </li><li>当Solaris定量PCR试剂后，储存在-20 ° C，直到他们准备使用。应收到以下试剂：一个20X的解决方案，其中包含两个引物在800 nm的每一个基因特异性探针在200纳米，色曲提供提供探针和引物，和Solaris的qPCR基因表达的2倍浓度的预混ENCE信息。至少12个月的Solaris qPCR的基因表达分析稳定。避免重复冷冻解冻。 </li></ol> 3。 qPCR的设置<ol><li>开始与基因的qPCR协议。应使用离心柱方法提取总RNA。建议的Thermo Scientific Verso公司cDNA合成试剂盒进行反转录生成的cDNA。 </li><li>解冻的引物，探针，并掌握冰混合准备的qPCR。另外手头上有PCR级水和白色不透明的qPCR板，它可以提高实时PCR的敏感性。 </li><li>主结构包含在内的所有实时PCR的组成部分：1）热启动DNA聚合酶，Thermoprime DNA聚合酶的热启动版本。这种类型的聚合酶是用来防止非特异性扩增反应的成立过程中和需要激活步骤在95 ° C 15分钟。 2）在Solaris预混dNTPs，dTTP的替代的dUTP最大限度地扩增效率3）专有反应​​缓冲液，优化工作与Solaris引物/探针检测。这个反应缓冲液含有蓝色染料增加试剂和塑料之间的对比。 4）ROX，如果您的qPCR仪器需要ROX。 </li><li>一旦解决方案已经解冻，混合弹管（不涡）。分拆下来，以防止试剂损失。 </li><li>下一步，准备一个15毫升无菌猎鹰管成立的感兴趣的基因，加上一个参照基因扩大取决于样本数，反应组合。这样三个复制可以为每个样品准备，包括无模板对照（NTC）和标准曲线的样品，如果将供分析用的标准曲线法，准备足够的。 </li><li>对于每个样品的96孔板上运行，混合以下内容：5μL2X的Solaris qPCR的预混，0.5μL的Solaris一套引物/探针（20X），PCR级水等，最终反应体积，一旦的cDNA，将25μL。对于每个样品384孔板上运行组合如下：12.5μL2X的Solaris qPCR的预混的Solaris一套引物/探针（20X），1.25μL，PCR级水等，最终反应体积，一旦基因添加10μL。 </li><li>使用多通道移液器，传输中，主机结构的钢板适当的水井。主结构中的蓝色染料，将有助于跟踪上的白色板吹打进展。然后，添加1-5μLcDNA模板（96孔板）或1-2μLcDNA模板（384孔板）。移液器上下搭配。板放置在离心机和自旋向下，以消除任何气泡，因为这些都会干扰与荧光读数。 </li><li>计划的热循环仪：必须激活这种酶与95 ° C，15分钟（1个周期）。 40周期95℃，15秒的变性，退火和延伸，在60 ° C为60秒。 </li><li>将在qPCR的仪器板。启动程序。 </li></ol>使用Solaris的代表qPCR的结果<img src="/files/ftp_upload/1700/1700fig1.jpg" alt="图1" /> 图1。PCR的效率和R2的值来判断的Solaris检测提供可靠的检测，即使在非常低的输入浓度。在ABI 7900HT使用的Solaris qPCR的基因表达检测仪器分别为F2RL1或CDC20，10个10倍稀释的合成，合成的RNA扩增序列或DNA扩增序列的cDNA扩增。日志规模的扩增曲线和标准曲线显示随着每次检测的性能，包括效率，R2的值，动态范围10 LOG10稀释，检测下限。 <img src="/files/ftp_upload/1700/1700fig2.jpg" alt="图2" /> 图2的Solaris分析给出一致的结果，甚至不同的研究人员和实验室之间。 100，10，1和0.1 nm的最终浓度的siRNA靶向ALDOA和PPIB，以及非针对控制（NTC），分别转染HeLa细胞。收获细胞总RNA隔离治疗后48小时。合成cDNA，使用Thermo Scientific的Verso公司cDNA合成试剂盒。在地理上分开的两个实验室使用罗氏的LightCycler 480（384孔）平台的Solaris qPCR的基因表达分析检测ALDOA，PPIB和GAPDH扩增的cDNA液。计算使用ΔΔCq法（归参照基因GAPDH的和负温度系数（NTC）处理的细胞）击倒。击倒相同的水平进行了论证研究人员在两个站点之间的靶基因。

这篇文章的作者是Thermo Scientific的员工。

Thermo Scientific的的Solaris qPCR实验提供了一个简单的，执行的qPCR分析方法的改进。这些套引物和探针使用高紧缩算法，结合MGB探针技术和Superbases，最大化的qPCR实验的保真度和鲁棒性。一个引物和探针组认识到所有已知的一个特定基因的剪接变异体。目标站点，只要有可能，包括跨越外显子区域，以确保未扩增，基因组DNA。这些特性使得可以同时运行众多的样品，大大减少了执行复杂的实验，所需的时间量。由于主结构是蓝色的，它有可能使用白色不透明板，这对提高定量PCR的灵敏度，而不会失去跟踪的吹打，的。 的Solaris定量PCR检测的设计特点，采取一切在一起，使这本小说的qPCR检测一个简单的，但具体和可靠的高性能的检测。

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

在Solaris qPCR的基因表达检测是一种新型的引物/探针设置，设计简化的qPCR的过程，同时保持检测的灵敏度和准确性。这些引物/探针集是预先设计&gt; 98％的人类和小鼠基因组和功能显着改善，从以前提供的技术。这些改进可能凭借Thermo Scientific的生物信息学专家的发展，一种新的设计方法。 几个方便的特点已被纳入到Solaris定量PCR检测，以简化过程进行实时定量PCR。首先，该协议是类似的普遍采用的替代品，所以在定量PCR所使用的方法很可能要熟悉。二，主结构是蓝色的，这使得设置的qPCR反应更容易追踪。三，热循环条件是所有检测（基因），从而有可能同时运行的许多样品和减少潜在的错误。最后，探针和引物序列信息提供，简化出版过程。 在这里，我们将演示如何使用产品搜索功能上的订购网站（www.thermo.com / Solaris）的发现和如何使用的Solaris试剂使用标准曲线法执行的qPCR GENEius获得适当的Solaris试剂。

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

的Solaris qPCR的基因表达分析小说预先设计的定量PCR引物/探针组合，旨在简化不牺牲检测的特异性和稳健性的qPCR过程。

实时定量PCR使用的Thermo Scientific的Solaris定量PCR检测

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 ...

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

Research

JoVE Journal

Biology

49.6K 次观看.  Thermo Scientific Solaris qPCR Products.  日本語 

视频: 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.

微量法测定蛋白浓度使用NanoDrop 2000C分光光度计

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

科研

生物学

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.

白蜡窄吉丁的基因表达分析（ Agrilus planipennis）使用实时定量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.

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

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微量核酸的定量

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.

确定基因表达概况 C。线虫使用微阵列和实时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, ...

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.

基因分型的植物和动物样品，恕不另行DNA的纯化

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

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

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.

定量，实时分析，利用分子信标病​​变特定的细胞裂解液中的碱基切除修复活动

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.

膜蛋白的实时测量:受体相互作用使用表面等离子体共振（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.

基于探针实时荧光定量PCR途径的小分子RNA定量检测

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

实时定量PCR使用的Thermo Scientific的Solaris定量PCR检测

副本

摘要

探索更多视频

此视频中的章节

微量法测定蛋白浓度使用NanoDrop 2000C分光光度计

白蜡窄吉丁的基因表达分析（ Agrilus planipennis）使用实时定量PCR

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

NanoDrop微量核酸的定量

确定基因表达概况 C。线虫使用微阵列和实时PCR

基因分型的植物和动物样品，恕不另行DNA的纯化

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

定量，实时分析，利用分子信标病变特定的细胞裂解液中的碱基切除修复活动

膜蛋白的实时测量:受体相互作用使用表面等离子体共振（SPR）

基于探针实时荧光定量PCR途径的小分子RNA定量检测

实时定量PCR使用的Thermo Scientific的Solaris定量PCR检测

副本

摘要

探索更多视频

此视频中的章节

微量法测定蛋白浓度使用NanoDrop 2000C分光光度计

白蜡窄吉丁的基因表达分析（ Agrilus planipennis）使用实时定量PCR

分析的预微RNA和小分子RNA采用实时定量PCR（定量PCR）阵列

NanoDrop微量核酸的定量

确定基因表达概况 C。线虫使用微阵列和实时PCR

基因分型的植物和动物样品，恕不另行DNA的纯化

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

定量，实时分析，利用分子信标病​​变特定的细胞裂解液中的碱基切除修复活动

膜蛋白的实时测量:受体相互作用使用表面等离子体共振（SPR）

基于探针实时荧光定量PCR途径的小分子RNA定量检测

定量，实时分析，利用分子信标病变特定的细胞裂解液中的碱基切除修复活动