MIQE Guidelines
qpcr步骤及原理
qPCR步骤及原理引言qPCR（quantitative polymerase chain reaction）是一种快速、敏感且特异性的检测DNA的方法。
它通过测量DNA模板的扩增过程中释放的荧光信号来定量检测目标DNA序列的数量。
本文将介绍qPCR的步骤及其原理。
步骤1. 样品准备在进行qPCR实验之前，需要准备样品。
通常情况下，样品可以是从生物体内提取的DNA，如细胞、组织、血液等。
样品的DNA含量应该在合适的浓度范围内，以确保qPCR的准确性和灵敏性。
2. 标准曲线标准曲线是进行定量PCR分析的关键。
它通过不同浓度的标准物质制备而成，通常是已知浓度的目标DNA序列。
标准曲线用于将实验测得的荧光信号与目标DNA的数量进行定量关联。
3. PCR反应体系准备PCR反应体系的准备十分重要，它包含了所有必要的试剂和反应物。
通常情况下，PCR反应体系包括DNA模板、引物、荧光探针（optional）、核酸酶、聚合酶和缓冲液。
荧光探针是一种具有荧光标记的DNA探针，用于增强PCR信号。
聚合酶是使PCR反应进行的关键酶。
4. qPCR循环程序qPCR的循环程序通常包括三个阶段：变性、退火和延伸。
在变性阶段，PCR管中的DNA双链被解链为两个单链。
然后，在退火阶段，引物结合到DNA模板的特定区域，并且荧光探针与目标DNA结合。
最后，在延伸阶段，聚合酶在引物的指导下合成新的DNA链。
5. 数据分析qPCR的荧光信号会随着PCR循环的进行而逐渐增加。
数据分析通常包括计算Ct值和绘制标准曲线。
Ct值是指荧光信号超过设定阈值所需的循环数，它与初始DNA模板的数量成正比。
原理qPCR基于传统的聚合酶链反应（PCR）技术，通过在DNA 扩增过程中测量荧光信号的释放来定量检测目标DNA序列的数量。
其主要原理可以概括为下列几个步骤：1.双链DNA解链：PCR反应体系中的DNA模板先经过高温变性处理，双链DNA解链为两个单链。
罗氏RNA提取试剂盒使用指南说明书
Step 1: Sample Preparation & Nucleic Acid IsolationFor great results, use (click product names to learn more):Roche High Pure RNA Isolation KitRoche High Pure FFPET RNA Isolation KitRoche High Pure miRNA Isolation KitRoche RealTime ready Cell Lysis KitFrom which source (animal, organ, tissue) does the examined material originally come from? Which volume or mass or cell number was used for nucleic acid preparation?My MIQE Guide*Empowering results that matter Sponsored by Roche Applied Science Experiment title:Performed by:Date:Institution:Experimental design: How did you choose and set up your study (number of treated samplesand controls)Handling: Which tools or methods were used to obtain and process the primary samples (e.g., micro-dissection, macrodissection)?Method of processing and preservation: How was the sample treated and stored?If frozen – how and how quickly?If fixed – with what, and how quickly?If stored for longer: how and how long? (especially for FFPE samples)Extraction method:Which kit or instrument was used to extract/isolate the DNA/RNA from the starting material? Roche High Pure RNA Isolation Kit, High Pure FFPET RNA Isolation Kit, High Pure miRNA Isolation Kit, RealTime ready Cell Lysis Kit, or other (Please specify)Was the vendor’s protocol modified (If Yes, when, and how? e.g. by using additives)Did you do a DNAse or RNAse treatment? (If Yes, when?)Did you check for nucleic acid purity and integrity? If Yes: By using which instrument and method? What was the resulting purity (A260/A280)? What was the resulting yield? If No: Why not?Did you check for the presence of PCR inhibitors? If Yes: By using what (e.g. Cq dilutions, spike or other (please specify)If No: Why not?Final storage solution (e.g., buffer, H2O) for the purified total RNA:Storage time and temperature of the purified total RNA before use in RT-qPCR:Step 2:Reverse TranscriptionFor optimal results, use:Roche Transcriptor First Strand cDNA Synthesis KitRoche Transcriptor Universal cDNA MasterAmount of RNA and reaction volume:Priming oligonucleotide (if using gene specific primers) and concentration: Reaction temperature and time:Manufacturer of reverse transcription reagent(s) and catalogue number(s): Reverse transcriptase type and used concentration:Storage conditions of cDNA:Step 3:PCR Amplification and AnalysisFor best results, use:LightCycler® 480 Probes MasterFastStart Essential DNA Probes MasterFastStart Universal Probe Master (Rox)Target sequence and amplicon information: Target gene database sequence accession number:Location of amplicon:Amplicon length:Result of in silico specificity screen (BLAST, etc.):Information on pseudogenes, retropseudogenes or other homologs: Secondary structure analysis of amplicon:Determined by which method?Location of each primer relative to exons or introns (if applicable): Targeted splice variants:RTPrimerDB Identification Numbers: Manufacturer of oligonucleotides: Purification method:For probe-based assays: Probe type:qPCR reaction conditionsReaction volume and amount of cDNA/DNA per reaction: Primer, (probe), Mg2+ and dNTP concentrations: Polymerase identity:Buffer/kit manufacturer and identity (e.g., catalog number)Manufacturer and catalog number of plates or tubes and catalog number:Complete thermocycling parameters:Reaction setup: Was it manual or robotic? If robotic: Using which robot?Equipment: Which Real-Time PCR instrument was used? (Which Roche LightCycler® System or other (please specify)?)Validation of qPCR runs:Are you running a multiplex assay? If yes, please describe efficiency and limit of detection foreach assay:How did you check for specificity of amplification for each target (e.g., on a gel, by sequencing, melt-ing curve analysis or digest):For SYBR Green I assays: Cq of the non-template control reaction:Standard curve characteristics (slope and y-intercept):How many replicates did you use to establish the standard curve?(xx replicates per standard concentration)What was the lower and the upper limit of the standard curve?PCR efficiency calculated from slope:Confidence interval for PCR efficiency or standard error:r2 of standard curve:Information on linear dynamic range:Cq variation at lower limit: Confidence intervals throughout range:Evidence for limit of detection:How many reactions per run were used for controls? (please specify positive and negative controls, controls without template and No RT controls, e.g. Positive controls: 3 reactions in 5 replicates per 96 well plate)Data analysis:Vendor software: Which software type, version and algorithm provided by the PCR machine supplier was used to analyze the data?Specialist software: Which (if any) additional software was used? Self-developed algorithms,or other (please specify)Normalisation: Which reference gene(s) were used to calculate the relative expression of the studied genes?What was the reason for choosing these particular genes?Which algorithm (e.g., geNorm, bestkeeper, normfinder) was used to normalize for reference gene(s)Which principle was used for Cq calling?What was the number and of biological replicates used?How was their concordance?How many technical replicates were used, and at which step (RT or qPCR)? What was the observed repeatability (intra-assay variation)?What was the observed reproducibility (inter-assay variation, %CV)The MIQE guidelines empower results that truly matter. And so does Roche.Visit to discover all the materials you need for truly remarkable research results.* modified based on the list in the original MIQE guidelines publication with permission of the MIQE authors.For life science research only. Not for use in diagnostic procedures. LIGHTCYCLER and FASTSTART are trademarks of Roche.All other product names and trademarks are the property of their respective owners. NOTICE: This product may be subject to certain use restrictions. Before using this product, please refer to the Online Technical Support page () and search under the product number or the product name, whether this product is subject to a license disclaimer containing use restrictions.Published byRoche Diagnostics GmbH Sandhofer Straße 116 68305 Mannheim Germany© 2013 Roche Diagnostics. 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BIORAD 荧光定量PCR手册
In this paper we demonstrate how to apply the MIQE guidelines (/miqe) to establish a solid experimental approach.
1. Experimental Design Proper experimental design is the key to any gene expression study. Since mRNA transcription can be sensitive to external stimuli that are unrelated to the processes studied, it is important to work under tightly controlled and welldefined conditions. Taking the time to define experimental procedures, control groups, type and number of replicates, experimental conditions, and sample handling methods within each group is essential to minimize variability (Table 1). Each of these parameters should be carefully recorded prior to conducting gene expression experiments to assure good biological reproducibility for published data.
pcr引物设计的基本理念 -回复
pcr引物设计的基本理念-回复PCR引物设计的基本理念PCR（聚合酶链反应）是一种常用的分子生物学技术，用于扩增DNA序列。
在PCR过程中，引物起着至关重要的作用，引物的设计质量直接影响PCR反应的效果。
因此，正确的PCR引物设计是成功进行PCR实验的基本前提。
本文将介绍PCR引物设计的基本理念，并分步回答如何设计合适的PCR 引物。
第一步：确认目标序列PCR实验通常是为了扩增某个特定的DNA序列，因此首先需要确定所需扩增的目标序列。
这个目标序列可以是基因片段、特定的DNA结构、启动子区域等。
在确认目标序列时，需要注意目标序列的长度和特异性，确保其能准确而特异地被引物扩增。
第二步：确定引物的长度引物通常由20到30个核苷酸组成，过短的引物可能不稳定，无法正确与模板DNA特异性结合，过长的引物则可能导致PCR产物过多的副产物。
因此，一般情况下引物长度约为18到24个核苷酸，适当选择引物长度有利于PCR的优化。
第三步：评估引物的理化性质引物的理化特性包括引物的GC含量、熔解温度（Tm）和自身引物结合能力。
高GC含量的引物更稳定，但也更难设计；Tm值表征了引物与模板DNA杂交的稳定性，通常理想的Tm值为50-65；自身引物结合能力的评估可以避免引物之间的非特异性结合。
多种软件可用于计算引物的理化性质，并为引物设计提供参考。
第四步：检查引物的特异性引物的特异性是PCR引物设计的关键点之一。
为了确保引物仅扩增目标序列，没有互补的序列存在于其他地方，可以使用引物设计软件进行特异性检查。
软件可以检查引物在基因组中的互补性和互补的副产物，以确保引物只扩增目标序列。
第五步：考虑引物间的配对引物在PCR反应中需要与目标序列的两端结合，因此两个引物（前向引物和反向引物）需要在目标序列上互补配对。
在设计引物时，需要确保两个引物的Tm值相近，以避免一个引物过早或过晚离开DNA链。
此外，还需要考虑引物之间的距离和可能的PCR产物长度，以确定引物的相对位置。
PCR程序两步法和三步法的区别是
PCR程序两步法和三步法的区别是引言聚合酶链反应（PCR）是一种在分子生物学领域广泛应用的技术，用于扩增DNA片段。
PCR可以使用两步法或三步法进行，这两种方法在程序设置和反应条件上有所区别。
本文将探讨PCR程序两步法和三步法之间的区别。
PCR的原理在介绍两步法和三步法之前，我们先来了解PCR的基本原理。
PCR的反应体系中包含待扩增的DNA模板，引物（primer），DNA聚合酶，反应缓冲液和dNTPs（脱氧核苷酸）。
通过一系列温度循环，PCR使DNA模板的目标区域反复复制，产生大量的目标DNA片段。
PCR程序的两步法两步法是最早采用的PCR程序方法之一，它包括三个主要步骤：变性、退火和延伸。
其中，变性步骤将高温（通常为94°C）用于使DNA模板两条链分离，即变性为两条单链。
接下来是退火步骤，将温度降低至引物能与目标DNA序列互补匹配的温度。
在这个温度下，引物与单链DNA发生互补结合。
最后是延伸步骤，将温度升高至DNA聚合酶的最适工作温度，使得DNA聚合酶能够从引物的3’端开始合成新的DNA链。
这样，PCR程序的一个循环就完成了。
两步法的优点是操作简便、反应时间相对较短，适用于一些简单的PCR反应。
然而，由于变性和延伸是在同一温度下进行，引物可能在变性温度下结合到非特异性的DNA序列上。
这会导致非特异性产物的形成，并可能影响PCR的结果。
PCR程序的三步法为了提高PCR的特异性，研究人员开发了三步法。
三步法在两步法的基础上增加了一个退火步骤，具体分为变性、退火和延伸三个步骤。
与两步法不同的是，退火步骤的温度通常要高于引物与目标DNA互补的温度。
这样可以使引物只与特异性的DNA序列结合，进一步提高PCR的特异性。
三步法相对于两步法来说，能够减少非特异性产物的产生，提高PCR的准确性和特异性。
它在一些复杂的PCR反应中表现良好，特别是在需要区分高度相似的DNA序列时。
结论PCR程序的两步法和三步法是在PCR技术发展过程中出现的两种主要方法。
wb蛋白提取步骤
wb蛋白提取步骤WB蛋白提取步骤引言：WB蛋白提取是一种常用的实验技术，用于研究蛋白质的表达及其相互作用。
本文将详细介绍WB蛋白提取的步骤，包括细胞裂解、蛋白质提取、浓缩和检测等关键步骤。
一、细胞裂解细胞裂解是WB蛋白提取的第一步，旨在破坏细胞膜、细胞核膜以及其他细胞组分，释放出目标蛋白质。
常用的细胞裂解方法有机械法、化学法和生物学方法等。
其中，最常用的方法是使用细胞裂解缓冲液，通过冷冻-解冻或超声波等方式破坏细胞结构。
二、蛋白质提取蛋白质提取是WB蛋白提取的关键步骤之一。
在细胞裂解后，目标蛋白质以及其他细胞组分被释放到裂解液中。
为了提取目标蛋白质，需要将裂解液进行离心，分离出上清液。
上清液中含有目标蛋白质，可以用于后续的蛋白质浓缩和检测。
三、蛋白质浓缩蛋白质浓缩是WB蛋白提取的重要步骤之一，旨在提高目标蛋白质的浓度，便于后续的蛋白质检测。
常用的蛋白质浓缩方法有醋酸沉淀法、酒精沉淀法和尿素沉淀法等。
在浓缩过程中需要注意控制温度和pH值，以避免蛋白质的降解和聚集。
四、蛋白质检测蛋白质检测是WB蛋白提取的最后一步，用于确定提取的蛋白质是否含有目标蛋白以及其相对丰度。
常用的蛋白质检测方法有SDS-PAGE和免疫印迹等。
其中，SDS-PAGE是一种分离蛋白质的电泳方法，可以根据蛋白质的分子量将其分离成不同的条带；免疫印迹则是通过特异性抗体与目标蛋白质结合，然后使用酶标记的二抗进行检测。
总结：WB蛋白提取是一种重要的实验技术，用于研究蛋白质的表达及其相互作用。
其步骤包括细胞裂解、蛋白质提取、蛋白质浓缩和蛋白质检测等。
通过合理选择和操作这些步骤，可以获得高质量的蛋白提取物，为后续的蛋白质研究提供可靠的基础。
参考文献：[1] Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611-622.[2] Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A.1979;76(9):4350-4354.[3] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, 1989.。
QuantStudio 3和QuantStudio 5实时PCR系统使用说明书
QuantStudio 3 and QuantStudio 5 Real-Time PCR SystemsConnect to your data anytime, anywhereThe modern laboratory is undergoing a technological revolution. Today’s scientific breakthroughs emerge in a context of unparalleled connectivity. Lab instruments not only are more compact, automated, powerful, and accessible, but also can store and can connect to platformsthat can share vast amounts of data, facilitating scientific advances through global collaboration.As your trusted partner at the leading edge of this era of innovation, we’ve developed the Applied Biosystems™QuantStudio™ 3 and 5 Real-Time PCR Systems. These high-performance benchtop instruments allow you to remotely monitor your runs, as well as easily access and securely share results with colleagues anywhere, anytime when connected to Thermo Fisher Cloud. With your data always within reach and shareable, the answers shaping the future of science are never far away.Connect with your future234•Q uickly share data setsand protocols online•S end large files securelyaround campus or aroundthe world•I ntegrate and analyzemultiple data sets anddata types into oneproject•A ccess experiment runsfrom any location, anytime,with remote monitoring•W i-Fi–enabled connectivity•U tilize portable devices to quicklyanalyze data when you need to* With Internet access and ThermoFisher ConnectInteractivityCollaboration* Accessibility*• Interactive touch screen•R un and edit directly from touch screen•E asy and intuitive interfaceThese instruments provide our latest advancements in touch-screen usability, allowing you to stay connectedto your data easily. They’re designed for both new and experienced users who need simple and affordable real-time PCR systems without compromising performance or quality.Access, analyze, and share data anytime, anywhere—Remotely monitor your runs, analyze sophisticated data sets in minutes, store data in a secure space, and share results online with colleagues across institutions and around the world, with Web browser–based software. Monitor your runs in real time from mobile devices through the Instrument Connect mobile app.Obtain results you can trust—Detect differences in target quantity as small as 1.5-fold in singleplex reactions, and obtain 10 logs of linear dynamic range.Establish standard operating procedures and compliance with ease—Locked protocol templates,in-run quality control (QC) feedback, and QC traceabilityof consumables offer greater control of experimental data. Real-time data mark-up language (RDML) export is available for compatibility with MIQE guidelines.Helps save valuable time—3 or 6 independent temperature zones for flexibility to run multiple experiments simultaneously. Fast thermal cycling is also available, enabling results in less than 30 minutes.Get started quickly—Instrument is factory-calibratedfor optical and thermal accuracy, quick installation, and immediate use.Skip the learning curve—With preoptimized protocol templates, training is minimized for new users, allowing you to focus on your research.Maximize benchtop space—Compact instrument can be configured as a stand-alone unit or with a computer to fit most laboratory needs.Get a premium instrument at an affordable price—Innovation doesn’t have to come at a premium price.Get the state-of-the-art Applied Biosystems functionality and industrial design that you’ve come to know, with the QuantStudio family of instruments.Discover the QuantStudio 3 and 5 Real-Time PCR SystemsThe QuantStudio 3 and 5 Real-Time PCR Systems are the latest additions to our family of QuantStudio systems.56• Interactive touch-screen interface and simplified Applied Biosystems TM QuantStudio ™ Design and Analysis Software make it easy to get started and stay organized• Easily identifiable icons guide you through the workflow to set up runs and analyze experiments• Graphical interface allows easy editing of experimental conditions (Figure 1A)• Interactive touch screen allows you to manipulate view to a particular graph or data point (Figure 1B)• Option to pause a real-time PCR run on demand• Preoptimized protocol templates allow quick selection of default protocols for standard applications • L ocked workflow feature allows for experimental consistency in tightly controlled environments InteractivitySimple, intuitive software—at your fingertipsFigure 1. Graphical interface allows (A ) easy editing of thermal cycling conditions and viewing of plate layout, as well as (B) viewing of amplification plotsand drilling down to a subset of sample wells.BA7Web-based or online:• Web browser–based system configuration with PC or Mac ™ computers• Streamlined software for improved usability and analysis response time• E nables secure access of your data when and where you want it• No software to install, no additional fees, and no versions to update• Monitor and check instrument status• Real-time run monitoring with Instrument Connect mobile appDesktop:• Simple co-located computer system configuration • Streamlined software for improved usability and analysis response timeFor more information about the Thermo Fisher Connect platform and data security, go to /connectTwo ways to access QuantStudio Design and Analysis SoftwareAccess with Web browser–based software Co-locate with computer8Applied Biosystems ™ Analysis Modules are innovative cloud-based data analysis applications that bring together multiple data sets in one convenient place, and render them in stunning data visualizations for enhanced analysis and insights.CollaborationFast and powerful secondary analysis software to extract and share resultsAnywhere, anytime accessAccess your data with a compatible browser on any device. Eachregistered user has a PIN-protected account on Thermo Fisher Connect.Fast and powerful analysisAnalysis speeds up to 10 times faster than our desktop software version, to help analyze more data and gain insights more quickly than before using Thermo Fisher Connect.Easy to useOne-click quality checks and comparisons between different visualizations, for simple and convenient data analysis.Integrated analysis solution Integrate your experiments into a single project—analyze various groups of data, such as time course experiments or cell line comparisons, and pick ideal settings to easily compare data.Superior securityPowered by Amazon Web Services ™, the Thermo Fisher Connect platform helps protect your data in a highly secure environment using 256-bit encryption and physical security measures.MIQE guideline supportThe instrument software allows users to save predefined analysis settings for auto-exporting run data into their format of choice, including RDML (real-time data mark-up language, compliant withMIQE guidelines) export format.9Figure 3. Relative quantification module for gene expression analysis. With this module, you can customize groupings of data within projects for a thorough comparison of data. The module also includes integrated correlation,volcano, and cluster plot analysis, with the ability to drill down to amplification plots.Figure 2. Absolute quantification module for gene expression analysis. The module enables analysis of genes of interest with the use of a standard curve. Additional flexibility is achieved by importing standard curves from other experiments.Figure 4. Genotyping analysis module. This module expands on existing Applied Biosystems ™ TaqMan ® Genotyper ™ software with improved visuals and integrated traces of allelic discrimination plots. The module allows for thorough quality control of SNP assays to accurately reflect true signals vs. background noise.Absolute quantificationRelative quantificationGenotypingThe Applied Biosystems Analysis Modules include:10Utilizing proven Applied Biosystems ™ OptiFlex ™ technology and VeriFlex ™ Blocks, QuantStudio 3 and 5 systems offerimproved data accuracy and sensitivity for a broad range of genomic applications, such as analyses of gene expression, microRNAs and noncoding RNAs, SNP genotyping, copy number variation, mutation detection, drug metabolism enzymes, and protein expression.Generate high-quality data for a variety of applicationsPerformance you can trustFigure 5. The QuantStudio 3 and 5 systems provide sensitive detection and high-confidence target discrimination down to 1.5-folddifferences. (A ) Amplification plots for 1.5-fold dilutions of a KAZ plasmid amplified with Applied Biosystems ™ PE2 TaqMan ® Assay under Fast run conditions using TaqMan ® Fast Advanced Master Mix. Quantities assayed, and C t (SD): 1,000 copies, 27.9 (0.063); 1,500 copies, 27.4 (0.059); 3,000 copies, 26.4 (0.060); 4,500 copies, 25.8 (0.047); 6,667 copies, 25.2 (0.049); 10,000 copies, 24.5 (0.041). NTC = no-template control. (B ) Standard curve generated from the C t values.BAFigure 6. Real-time PCR reproducibility. This plot shows results from amplification of KAZ target plasmid DNA in 10-fold dilutions using the 96-well block. The data show highly reproducible results over 10 logs of inputtemplate amount, illustrating the broad linear dynamic range of the system.Precise quantification with 1.5-fold discriminationExcellent reproducibility and 10-log dynamic range11The QuantStudio 3 and 5 systems support probe-based assays as well as intercalating dyes. TaqMan ® probe-based assays, developed with powerful algorithms and optimized master mixes, enable outstanding specificity and sensitivity. Applied Biosystems ™ SYBR ™ Green chemistry is an economical alternative for target identification or initial screening assays. The QuantStudio 3 system has 4 filters calibrated for FAM ™/SYBR Green, VIC ™/JOE ™, NED ™/TAMRA ™, and ROX ™ dyes. The QuantStudio 5 System offers 96- w ell and 384-well format options, allowing for a broader range of detection chemistries and assay multiplexing. The 96-well format has 6 excitation filters (450–680 nm) and 6 emission filters (500–730 nm), and the 384-well format has 5 excitation filters (450–650 nm) and 5 emission filters (500–700 nm).Generate high-quality data for a variety of applicationsAssay flexibility to support your applicationGenotyping analysisFigure 9. Allelic discrimination plot with traces using real-time PCR data. Cluster plot of 44 gDNA samples and 4 no-template controls (NTCs) genotyped using Applied Biosystems ™ T aqMan ® SNP Genotyping Assay C_29086771_20, with both PCR and allelic discrimination performed on the QuantStudio 5 Real-Time PCR System. The novel use of real-time PCR data to plot SNP cluster progress aids in calling ambiguous samples and reduces run times by displaying the optimal number of cycles necessary for maximum cluster separation.Figure 8. Multiplex reaction with 4 targets plus passive reference.Whole-plate amplification plots of 96 replicates of cDNA made from universal human RNA (UHR) amplified under Fast run conditions using AppliedBiosystems ™ TaqMan ® Multiplex Master Mix with Mustang Purple ™ passive reference dye. Targets and labels: FZD1 labeled with FAM dye, APOE labeled with VIC dye, CD44 labeled with ABY ™ dye, GAPDH labeled with JUN ™ dye.Melt curve analysisMultiplex gene expressionFigure 7. Melt curve analysis using the online version of the software. In this experiment, 96 replicates of human genomic DNA were amplified using Applied Biosystems ™ SYBR ™ Select Master Mix with primers for RNase P followed by a dissociation step. The reactions were run under Fast run conditions, showing C t uniformity with a mean of 25.7 (SD 0.077), and thermal uniformity as measured by the derivative peak with a melting temperature (T m ) of 84.17°C (SD 0.07°C).For more information about TaqMan Assays and formats, go to/taqmanTechnical specificationsQuantStudio 3QuantStudio 59696 or 3840.1 mL block: 10–30 μL 0.2 mL block: 10–100 μL 96-well 0.1 mL block: 10–30 μL 96-well 0.2 mL block: 10–100 μL 384-well: 5–20 μL27 cm x 50 cm x 40 cm27 cm x 50 cm x 40 cmBright white LED Bright white LED4 coupled filters96-well: 6 decoupled filters384-well: 5 coupled filters450–600 nm/500–640 nm96-well: 450–680 nm/500–730 nm384-well: 450–650 nm/500–700 nm Multiplexing Up to 4 targets96-well: up to 6 targets384-well: up to 5 targetsOptional OptionalPeltier Peltier3 VeriFlex zones96-well: 6 VeriFlex zones384-well: NA0.2 mL block: 6.5°C/sec 0.1 mL block: 9.0°C/sec 0.2 mL block: 6.5°C/sec 0.1 mL block: 9.0°C384-well block: 6.0°C/sec3.66°C/sec 3.66°C/sec0.4°C0.4°C0.25°C0.25°CRun time<30-minute runs96-well block: <30-minute runs384-well block: <35-minute runsFAM/SYBR Green, VIC/JOE/HEX/TET, ABY/NED/TAMRA/Cy®3, JUN, ROX/ Texas Red™FAM/SYBR Green, VIC/JOE/HEX/TET, ABY/NED/TAMRA/Cy3, JUN, ROX/ Texas Red, Mustang Purple, Cy®5/LIZ™, Cy®5.5Fast/standard Fast/standardNo Yes, with no additional fees 1 copy 1 copySensitivity Detect differences as small as 1.5-fold in target quantities in singleplexreactions Detect differences as small as 1.5-fold in target quantities in singleplex reactions1213Service and support to help meet your changing needsSmartStart orientationEvery QuantStudio 3 and QuantStudio 5 system includes a SmartStart orientation to get you up and running quickly in your lab. The orientation includes basic qPCR familiarization and setup with both Thermo Fisher Connect and online Instrument Management. QuantStudio 5 system owners receive a personalized qPCR application training.Online instrument management Sign in to your account to access the award-winning* free online Instrument Management** tool that enables faster responses to requests for service or service quotes, plus fast connection to key instrument and service information.Comprehensive instrument warrantyOur factory-trained and certified field service engineers (FSEs) are focused on delivering the highest-quality workmanship. During the warranty period, all qualifying repairs, including engineer time and travel, are covered.Flexible service plansChoose from a variety of serviceoptions that balance your budget,productivity, uptime, and regulatoryrequirements. Plans start with themost basic repair models and scale topremium offerings including advancedsupport and compliance services.On-site service plans are optimal forlabs that have time-sensitive workand need to get their instrumentback online quickly. These plansinclude guaranteed response timesin most regions, scheduled plannedmaintenance, and automatic softwareupdates. The AB Repair Center plan isa cost-effective choice for customerswho can allow their instrument to besent away for repair—this plan providesa loaner instrument so that customerscan maintain productivity while theirinstrument is being repaired.Professional servicesOur services are designed to helpyou balance business and regulatoryrequirements—from risk assessment,hardware/software qualification, fullsystem validation, and LIMS interfacingservices to data storage and backupsolutions. We partner with you to helpmitigate regulatory risks, get yourprocesses up and running, and helpensure data integrity across your lab.Training coursesOur application and instrument trainingprograms are led by scientists whoaim to enhance your workday throughexperimental design best practices,workflow training, and instrumenttroubleshooting. Hands-on classes areavailable at our Thermo Fisher Scientifictraining centers or in your lab.Technical supportIf you have questions aboutproduct selection or use, assay orexperimental design, data analysis, ortroubleshooting, contact our team oftechnical support scientists or accessour online product and applicationsupport tools.Financing optionsIf you’re looking for acceleratedreturn on investment, technologyprotection, or cash flow management,our innovative financing options canhelp meet your company’s budgetaryneeds and bottom-line goals. Contactyour local sales representative formore details.For a full schedule of courses, including self-paced online classes, go to /training * 2012 Oracle Fusion Middleware Innovation Award.** Online Instrument Management tool not available in all regions.14Service plans at a glanceTarget 2 business days*Guaranteed 2business days*Guaranteednext businessday*✓✓✓✓✓✓✓✓✓✓✓10% discountoptional add-on in selectedregions✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓✓Response times vary by region.15Ordering informationHow to reach usTo find your order support or technical support team, go to /contactusFor product FAQs, protocols, training courses, and webinars, go to /technicalresources For more info, go to/quantstudio3-5For Research Use Only. Not for use in diagnostic procedures. © 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarksare the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of RocheMolecular Systems, Inc., used under permission and license. Cy is a registered trademark of GE Healthcare. Mac is a trademark of Apple Inc.Amazon Web Services is a trademark of Amazon Technologies, Inc. Oracle is a trademark of Oracle International Corporation.COL03261 0117。
MIQE指南范文
MIQE指南范文MIQE（Minimum Information for Publication of Quantitative Real-Time PCR Experiments）指南是一个旨在提高实时定量PCR（qPCR）实验的透明度和可重复性的指南。
下面是一篇关于MIQE指南的范文，字数超过1200字：Abstract:Introduction:Material and Methods:1. Sample Collection: Tissue samples from different organs were collected from ten healthy individuals. The samples were immediately frozen in liquid nitrogen to preserve RNA integrity.2. RNA Extraction: Total RNA was extracted from the tissue samples using the TRIzol reagent following the manufacturer's instructions. RNA concentration and quality were determinedusing a spectrophotometer.3. cDNA Synthesis: RNA samples were treated with DNase I to remove genomic DNA contamination. cDNA was synthesized using the SuperScript III Reverse Transcriptase kit following the manufacturer's protocol.5. qPCR Assay: qPCR amplifications were performed using the SYBR Green PCR Master Mix and the Applied Biosystems 7500 Real-Time PCR System. The cycling conditions consisted of an initialdenaturation at 95°C for 10 minut es, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.6. Data Analysis: The cycle threshold (Ct) values were determined using the Applied Biosystems software. The relative expression levels of Gene X were calculated using the 2^-ΔΔCt method, with GAPDH as the reference gene.Results:1. RNA Concentration and Quality: The concentration and purity of the RNA samples were assessed using a spectrophotometer. The samples exhibited an A260/A280 ratioof >1.8, indicating high RNA purity.2. Primer Specificity and Efficiency: The primer specificity was confirmed by the presence of a single peak in the melting curve analysis and the absence of non-specific amplification products. The primer efficiency was determined using a standard curve, which exhibited a slope of -3.32, corresponding to an efficiency of 97.9%.Discussion:Conclusion:In conclusion, following the MIQE guidelines is crucial for conducting accurate and reproducible qPCR experiments. This study demonstrated the successful application of the MIQE guidelines in investigating the expression of Gene X in varioustissues and conditions. By adhering to these guidelines, we were able to obtain reliable data, contributing to a better understanding of gene expression regulation.。
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40 cDNA Standard Total RNA Standard
•
35
T
30
25
20 cDNA -3.394 0.999 38.91 97.1% total -3.382 0.999 38.09 97.6%
Primer B
Forward Primer
Reverse Primer A
1
110
/pcr
Amplicon Secondary Structures
AMPLIFICATION
/mfold/applications
/pcr
AMPLIFICATION
Nucleic Acid Extraction （核酸提 取）
/pcr
RNA 纯度和完整性
AMPLIFICATION
Experion Virtual Gel
L C 3’ 5’ 10’ 15’ 1h 2h 4h
/pcr
AMPLIFICATION
/pcr
RNA analysis
AMPLIFICATION
• Experion™ system
Good Quality
Bad Quality
/pcr
Impact of RNA quality
Experiment
Control - NTC用来验证实验材料是否具有污染。 - NRT是未经反转录的RNA作为阴性对照。对 gDNA残留的控制。
/pcr
NTC/NRT
Sample treatment/preparation
AMPLIFICATION
• Replicates
/pcr
qPCR Target Information
AMPLIFICATION
CATION
http://www.ncbi.nlm.nih. gov/BLAST/Blast.cgi
/pcr
/pcr
AMPLIFICATION
A real case
- Gene Expression results submitted to Plant Cell - dtdt Ct, but … - No proof of 100% efficiencies from both target gene and reference gene - Requested to provide supplementary data 启示: 实验以前应该先研究一下这个指南
AMPLIFICATION
What is MIQE? It’s a Checklist (检查清单) !
– Experimental Design （实验设计） – Sample Information （样本信息 ） – Nucleic Acid Extraction （核酸提取） – Reverse Transcription （反转录） – qPCR Target Information – （目的基因信息） – qPCR Oligonucleotides – （qPCR 寡核苷酸） – qPCR Protocol （qPCR 程序） – qPCR Validation （qPCR验证） – Data Analysis （数据分析）
不管target gene的丰度如何、起始的RNA输入量如何，反转录的效率都是 一致的 确保cDNA的差异能够真实反映 RNA i.e. 基因表达的差异。 不 影响表达差异的判定。
/pcr
AMPLIFICATION
反转录效率一致性可以通过预 实验来判定
实验: 梯度稀释RNA及cDNA
8000 7000 5893 6000 5230 5000 4000 3000 2000 1000 18 0 21 37 128 741 361 1417 3402 2325 4693 6643
7236
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
编辑们犯难了“定量PCR数据可信吗？”
/pcr
What are the MIQE guidelines?
AMPLIFICATION
qPCR的国际标准：就评价qPCR实验和发表文章时所必需的实验 信息提出了最低限度的标准。
/pcr
MIQE 指南好处
AMPLIFICATION
•
规范专业术语和概念
– 术语: reference genes, qPCR, Cq (quantification cycle ) – 概念: sensitivity, specificity, accuracy, repeatability, reproducibility
/pcr
MIQE Guidelines
AMPLIFICATION
• • •
A Check list of 80+ items that describe the minimum information necessary to evaluate qPCR experiments Broken into essential (E) and desirable (D) categories of information Ideally the check list should be submitted with manuscripts and available online to researchers (RDML)
• • •
Web software provided by Steve Rozen and Whitehead Institute for Biomedical Research. /cgi-bin/primer3/primer3_www.cgi
AMPLIFICATION
qPCR Oligonucleotides （引物和探针）
/pcr
AMPLIFICATION
Real case 二级结构的影响及引物位置
Primer A
Primer A: = 66.3 % Primer B: = 95.8 %
Reverse Primer B
以Real-time PCR为关键词在 NCBI Pubmed 检索结果
/pcr
AMPLIFICATION
Questionable Data 问题数据
Slope : -1.365 Eff = 440.26% Slope : -2.276 Eff = 175.02%
/pcr
Corresponding Nanodrop Readings
Sample Control-no heat 3 min @ 90C 5 min @ 90C 10 min @ 90C 15 min @ 90C 1 hr @ 90C 2hr @ 90C 4 hr @ 90C Conc (ng/ul) 115 114 115 115 116 109 117 118 A260/280* 1.90 1.93 2.06 2.03 2.02 1.99 2.00 1.89 A260/230* 2.44 2.40 2.37 2.37 2.31 2.18 2.32 2.23
MIQE - 实时荧光定量PCR实验流程 及数据处理的国际化标准及语言规则
定量PCR发展及遇到问题
AMPLIFICATION
-
大量的科研工作使用定量PCR，每年产生大量的文章，但是
如何合理设计实验没有共识 如何描述实验结果没有共识 （gene表达差异的柱状图，etc） 投稿时提供什么实验信息没有共识 （自身说服力差、编审犯难、同行无法重复、出现大量错误 结果）
– Need for both technical replicates and Biological replicates. – Number of replicates will depend on level of differences that are being presented. – Lower expression genes tend to require more replicates to establish statistical validity of small differences.
Biological
Technical
/pcr
Sample Information （样本信息）
AMPLIFICATION
判定材料的质量
- 对样品材料的描述 - 肿瘤里边切下的肿瘤材料是否 带有健康组织，etc。 - 处理过程- 是否是新鲜材料 - 存储方式、时间 -医学里边，材料的固定和处理 方式
•
规范操作标准
– 样品获取、 处理和制备 – RNA 或 DNA质控 – 反转录 – qPCR 过程(酶,、耗材、 特异性、引物序列、 二级结构,、对照、校正) – 数据分析(均一化, 生物重复和技术重复)
– 评审可以更好的评估研究者所用的实验方案的有效性 – 确保文献的完整性和可靠性
/pcr
AMPLIFICATION
Retracted Paper 召回的文章
/pcr
数据处理难点环节
AMPLIFICATION
• • • • •
相关性（扩增效率） 准确性（内参基因，扩增子二级结构） 重复性与重现性 数据分析（均一化，变异性分析） 统计分析
大量无统计学意义和相互矛盾结果的发表
/pcr
Experimental Design 实验设计
AMPLIFICATION
- 实验样品的分类，处理和对照都要具备并明确定义 - 重复数
/pcr