The 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. All rights reserved.*********** 1012。
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.
MIQE指南
Bulletin CN 5859
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Fleige S and Pfaffl MW (2006). RNA integrity and the effect on the real-time qRTPCR performance. Mol Aspects Med 27, 126-139.
Garson JA et al. (2009). Unreliable real-time PCR analysis of human endogenous retrovirus-w (HERV-W) RNA expression and DNA copy number in multiple sclerosis. AIDS Res Hum Retroviruses 25, 377-378; author reply 379-381.
Bio-Rad’s real-time thermal cyclers are licensed real-time thermal cyclers under Applera’s United States Patent 6,814,934 B1 for use in research, human in vitro diagnostics, and all other fields except veterinary diagnostics. This product is covered by one or more of the following U.S. patents or their foreign counterparts owned by Eppendorf AG: U.S. Patent Nos. 6,767,512 and 7,074,367.
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.。
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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The MIQE Guidelines:M inimum I nformation for Publication of Q uantitativeReal-Time PCR E xperimentsStephen A.Bustin,1*Vladimir Benes,2Jeremy A.Garson,3,4Jan Hellemans,5Jim Huggett,6Mikael Kubista,7,8Reinhold Mueller,9Tania Nolan,10Michael W.Pfaffl,11Gregory L.Shipley,12Jo Vandesompele,5and Carl T.Wittwer 13,14BACKGROUND :Currently,a lack of consensus exists onhow best to perform and interpret quantitative real-time PCR (qPCR)experiments.The problem is exac-erbated by a lack of sufficient experimental detail in many publications,which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments.CONTENT :The Minimum Information for Publicationof Quantitative Real-Time PCR Experiments (MIQE)guidelines target the reliability of results to help ensure the integrity of the scientific literature,promote con-sistency between laboratories,and increase experimen-tal transparency.MIQE is a set of guidelines that de-scribe the minimum information necessary for evaluating qPCR experiments.Included is a checklist to accompany the initial submission of a manuscript to the publisher.By providing all relevant experimental conditions and assay characteristics,reviewers can as-sess the validity of the protocols used.Full disclosure of all reagents,sequences,and analysis methods is neces-sary to enable other investigators to reproduce results.MIQE details should be published either in abbreviated form or as an online supplement.SUMMARY :Following these guidelines will encouragebetter experimental practice,allowing more reliable and unequivocal interpretation of qPCR results.©2009American Association for Clinical ChemistryThe fluorescence-based quantitative real-time PCR (qPCR)15(1–3),with its capacity to detect and mea-sure minute amounts of nucleic acids in a wide range of samples from numerous sources,is the enabling tech-nology par excellence of molecular diagnostics,life sci-ences,agriculture,and medicine (4,5).Its conceptual and practical simplicity,together with its combination of speed,sensitivity,and specificity in a homogeneous assay,have made it the touchstone for nucleic acid quantification.In addition to its use as a research tool,many diagnostic applications have been developed,in-cluding microbial quantification,gene dosage determi-nation,identification of transgenes in genetically mod-ified foods,risk assessment of cancer recurrence,applications for forensicuse (6–11).This popularity is reflected in the number of publications reporting qPCR data,which invariably use diverse reagents,protocols,methods,and reporting formats.This lack of consensus on how best to perform qPCR periments has the adverse consequence of ating a string of serious shortcomings that encumber its status as an independent yardstick (12).Techni-cal deficiencies that affect assay performance include the following:(a )inadequate sample storage,prep-aration,and nucleic acid quality,yielding highly variable results;(b )poor choice of reverse-transcription primers and primers and probes for the PCR,leading to inefficient and less-than-robust assay performance;1Centre for Academic Surgery,Institute of Cell and Molecular Science,Barts and the London School of Medicine and Dentistry,London,UK;2Genomics Core Facility,EMBL Heidelberg,Heidelberg,Germany;3Centre for Virology,Depart-ment of Infection,University College London,London,UK;4Department of Virology,UCL Hospitals NHS Foundation Trust,London,UK;5Center for Medical Genetics,Ghent University Hospital,Ghent,Belgium;6Centre for Infectious Diseases,University College London,London,UK;7TATAA Biocenter,Go ¨teborg,Sweden;8Institute of Biotechnology AS CR,Prague,Czech Republic;9Seque-nom,San Diego,California,USA;10Sigma–Aldrich,Haverhill,UK;11Physiology Weihenstephan,Technical University Munich,Freising,Germany;12Quantita-tive Genomics Core Laboratory,Department of Integrative Biology and Phar-macology,University of Texas Health Science Center,Houston,Texas,USA;13Department of Pathology,University of Utah,Salt Lake City,Utah,USA;14ARUP Institute for Clinical and Experimental Pathology,Salt Lake City,Utah,USA.*Address correspondence to this author at:3rd Floor Alexandra Wing,The Royal London Hospital,London E11BB,UK.Fax ϩ44-(0)20-73777283;e-mail ******************.uk.Received October 20,2008;accepted January 27,2009.Previously published online at DOI:10.1373/clinchem.2008.11279715Nonstandard abbreviations:qPCR,quantitative real-time PCR;MIQE,Minimum Information for Publication of Quantitative Real-Time PCR Experiments;RT-qPCR,reverse transcription–qPCR;FRET,fluorescence resonance energy trans-fer;C q ,quantification cycle,previously known as the threshold cycle (C t ),crossing point (C p ),or take-off point (TOP);RDML,Real-Time PCR Data Markup Language;LOD,limit of detection;NTC,no-template control.Clinical Chemistry 55:4000–000(2009)Reviews1/cgi/doi/10.1373/clinchem.2008.112797The latest version is at Papers in Press. Published February 26, 2009 as doi:10.1373/clinchem.2008.112797Copyright (C) 2009 by The American Association for Clinical Chemistry本页已使用福昕阅读器进行编辑。