Bio-rad实实时荧光定量时荧光定量PCR仪仪BIO-RAD BIO-RAD Gene Expression DivisionGene Expression DivisionOutlineReal Time PCR and Conventional PCRIntroduction to Quantitative PCRCt value and Real-Time Quantitative PCR TheoryLaboratory TechniquePrimer Design for Real-Time PCRReal Time PCR and Conventional PCR技术背景本来，本来，PCRPCR技术是为了将样本中微量的技术是为了将样本中微量的DNADNA模版放大模版放大以便研究模版特性以便研究模版特性随着研究的深入，需要了解样本中基因的表达模式随着研究的深入，需要了解样本中基因的表达模式与疾病的关系，这就要了解标本中的与疾病的关系，这就要了解标本中的DNADNA原始拷贝原始拷贝数。数。DenaturationPrimer AnnealingElongation53535353535355TaqTaqRepeat常规常规 PCR ProcessIn theory, product accumulation is proportional to 2n, where n is the number of amplification cycle repeatsnA linear increase follows exponentialnEventually plateausCycle #TheoreticalReal LifeLog Target DNAReality vs. TheoryAmplification is exponential, but the exponential increase is limited:常规常规PCR方法的局限性分析：方法的局限性分析：无法对起始模板准确定量无法对起始模板准确定量, ,只能对终产物进只能对终产物进行分析行分析对终产物分析，受对终产物分析，受PCRPCR过程平台效应的干扰，过程平台效应的干扰，定量准确度难以提高（相对误差定量准确度难以提高（相对误差1000%1000%）；）；存在扩增产物的污染问题。存在扩增产物的污染问题。必须在扩增后用电泳方法分析必须在扩增后用电泳方法分析, ,费时费力而费时费力而且且EBEB有毒有毒无法对扩增反应实时检测无法对扩增反应实时检测 C(t)s from 96 replicates are nearly identical, but the final fluorescence varies.CycleEnd PointPCR: Theory vs. Reality 实时定量实时定量PCR技术，是指在技术，是指在PCR反应体系中反应体系中加入加入荧光基团荧光基团，利用荧光信号积累实时监测整个，利用荧光信号积累实时监测整个PCR进程，使每一个循环变得进程，使每一个循环变得“可见可见”，最后通，最后通过过Ct值和标准曲线值和标准曲线对样品中的未知样品对样品中的未知样品 的起始浓的起始浓度进行定量的方法。度进行定量的方法。 Real-Time PCR Quantitative PCR 或或 Real time PCR 是确定样品中是确定样品中DNA (或或 cDNA) 拷贝拷贝数最敏感、最准确的方法数最敏感、最准确的方法Quantitative PCR /Real time PCR C(t)s from 96 replicates are nearly identical, but the final fluorescence varies.CycleEnd PointC(t)PCR: Theory vs RealityQuantitative information comes from monitoring the early stages of amplification.Cycle #Cycle #TheoreticalTheoreticalReal LifeReal LifeLog Target DNALog Target DNADetectorDetectorDetector定量的最佳时期定量的最佳时期定量的最佳时期定量的最佳时期常用的定量方法常用的定量方法相对定量：相对于另一参照样本的量；相对定量：相对于另一参照样本的量；绝对定量：用标准品作标准曲线来推算未知的绝对定量：用标准品作标准曲线来推算未知的样本的量。样本的量。Quantitative PCR和常规和常规PCR技术的区别技术的区别常规常规PCR是通过电泳对扩增反应的最终产物进是通过电泳对扩增反应的最终产物进行定性分析（行定性分析（定量不准确定量不准确）；）；Quantitative PCR是在是在PCR反应体系中加入荧反应体系中加入荧光基团，利用荧光信号积累实时监测整个光基团，利用荧光信号积累实时监测整个PCR进程，使每一个循环变得进程，使每一个循环变得“可见可见”，通过，通过Ct值值和标准曲线对样品中的和标准曲线对样品中的DNA (or cDNA) 的起始的起始浓度进行定量的方法（浓度进行定量的方法（准确定量准确定量） 。Introduction to Quantitative PCRPCR仪仪 + 监测、分析系统监测、分析系统 + 荧光标记荧光标记 采用半导体加热和制冷采用半导体加热和制冷采用半导体加热和制冷采用半导体加热和制冷加热速度加热速度加热速度加热速度 3.3C /3.3C /秒秒秒秒 ，制冷速度，制冷速度，制冷速度，制冷速度 2C/2C/秒秒秒秒升降温速度可调升降温速度可调升降温速度可调升降温速度可调控温准确性：控温准确性：控温准确性：控温准确性：0.30.3定量定量定量定量PCRPCRPCRPCR仪应该具备的特点：杰出的温度控制仪应该具备的特点：杰出的温度控制仪应该具备的特点：杰出的温度控制仪应该具备的特点：杰出的温度控制温度梯度温度梯度选择可以选择可以允许使用者在同一允许使用者在同一次扩增中优化复性次扩增中优化复性温度。温度。定量定量定量定量PCRPCRPCRPCR仪应该具备的特点：梯度仪应该具备的特点：梯度仪应该具备的特点：梯度仪应该具备的特点：梯度PCRPCRPCRPCR功能功能功能功能采用多点温控和传采用多点温控和传感技术，可以实现感技术，可以实现梯度梯度PCR功能功能采用采用0.2ml的离心管的离心管, 排管和排管和 96孔孔PCR反应板。反应板。 降低消耗品成降低消耗品成本。本。定量定量定量定量PCRPCR仪应该具备的特点：仪应该具备的特点：仪应该具备的特点：仪应该具备的特点：消耗品成本低，样品容量大消耗品成本低，样品容量大消耗品成本低，样品容量大消耗品成本低，样品容量大同时扩增和检测同时扩增和检测96个样品个样品Introduction to Quantitative PCRPCR仪仪 + 监测、分析系统监测、分析系统 + 荧光标记荧光标记 定量定量定量定量PCRPCRPCRPCR仪应该具备的特点：仪应该具备的特点：仪应该具备的特点：仪应该具备的特点： 先进的光路设计先进的光路设计先进的光路设计先进的光路设计-多孔样品同时检测多孔样品同时检测多孔样品同时检测多孔样品同时检测DetectorFluorescence DetectionEmissionFilterLight SourceExcitationFilter53FQTubulin53HQGapdH53TXQb b-actin53Cy5QCyclophilinMultiplexing-同时检测同时检测5色色荧光荧光CycleLog Relative FluorescenceTexas RedFAMHexCy5Multiplexing定量定量定量定量PCRPCRPCRPCR仪应该具备的特点：仪应该具备的特点：仪应该具备的特点：仪应该具备的特点：开放性的系统开放性的系统开放性的系统开放性的系统v可兼容的定量方法：可兼容的定量方法： SYBR Green I、Taqman探针、探针、Beacon探针、探针、FRET探针探针v可兼容的突变检测方法：可兼容的突变检测方法： 熔点曲线功能、熔点曲线功能、MGB探针探针v可兼容的试剂种类：可兼容的试剂种类： 所有国产与进口试剂所有国产与进口试剂 iQ5 Data Analysis Software iQ5 Data Analysis Software 定量定量定量定量PCRPCR仪应该具备的特点：仪应该具备的特点：仪应该具备的特点：仪应该具备的特点：分析软件功能强大简洁直观分析软件功能强大简洁直观分析软件功能强大简洁直观分析软件功能强大简洁直观点击您所需要的程序名称，点击您所需要的程序名称，该程序所包含的所有内容该程序所包含的所有内容都一目了然。方便您在众都一目了然。方便您在众多的程序中快速找到您所多的程序中快速找到您所需要的一个。需要的一个。黄色提示会警告黄色提示会警告您在编辑的程序您在编辑的程序中存在逻辑错误中存在逻辑错误的数据。您可重的数据。您可重新审查、修改。新审查、修改。在运行开始之前，警示在运行开始之前，警示框提示数据设置的残缺。框提示数据设置的残缺。异常数据或情况提示异常数据或情况提示异常数据或情况提示异常数据或情况提示标准曲线标准曲线定量计算定量计算自动数据分析自动数据分析自动数据分析自动数据分析多种报告单模式可供选择多种报告单模式可供选择多种报告单模式可供选择多种报告单模式可供选择-尤其重要尤其重要尤其重要尤其重要基因表达分析Introduction to Quantitative PCRPCR仪仪 + 监测、分析系统监测、分析系统 + 荧光标记荧光标记 l ll ll ll lDetection ChemistriesNon-specific DNA binding dyesSYBR Green ISYBR GoldEthidium Bromide Specific Hybridization ProbesCleavage Probes (TaqManTM)Molecular beaconsScorpionsTMAmplifluorTMLUXTMdual-oligo FRET pairsQuantitative PCR Detection ChemistriesDNA Binding Dyes35BDBD5BDBDBDExtension53535353Extension ContinuedApply ExcitationWavelength535355TaqTaq353TaqTaq55RepeatDNA binding dyesBDBDBDBDBDBDBDBDBDBDlllllDNA Binding Dyes Advantages!Inexpensive compared to hybridization probesNo additional design work than the primer used for PCR reaction However Not template specific, will bind ALL double stranded DNA inducing primer-dimer and unspecific amplicon formationMultiplex assays not possibleSYBR Green I ExperimentTypical “first step” experiment: Evaluate Primer SpecificityUsing Melt Curve Analysis Evaluate Primer Pair EfficienciesBy running serial dilutions of template as standards Identify Sub-Optimal aspects of assayOptimize further with thermal gradient, etc.Cleavage Probes (TaqManTM)53QFTaq5q3l l3535FQn n杂交探针的互补区在引物间杂交探针的互补区在引物间杂交探针的互补区在引物间杂交探针的互补区在引物间n n55端连有一个荧光端连有一个荧光端连有一个荧光端连有一个荧光reporterreporter, , 通常是荧光素通常是荧光素通常是荧光素通常是荧光素n n33端连有一个端连有一个端连有一个端连有一个QuencherQuencher, ,通常是通常是通常是通常是TAMRATAMRAn n荧光素被荧光素被荧光素被荧光素被488 488 nmnm光激发，发射光为光激发，发射光为光激发，发射光为光激发，发射光为520 520 nmnmn n520 nm 520 nm 光能激发光能激发光能激发光能激发TAMRATAMRA，发射光为发射光为发射光为发射光为570 570 nmnmTaqManTaqManCleavage-based assay:TaqManTM5533d.NTPsThermal Stable DNA PolymerasePrimers5353535353535353Add Master Mix and SampleDenaturation535353535353AnnealingReaction TubeTaql53RQProbe53RQ535353RQExtension Step531. Strand DisplacementTaq3QR5533QTaqR52. Cleavage3. PolymerizationComplete53QTaqR354. Detection533QTaqR5lRCleavage-based assay:TaqManTMCleavage Probes (TaqManTM)Advantages!Generates a robust cumulative fluorescence signalSimple to design and synthesize compared to other hybridization probes (i.e. beacons)Ideal approach for multiplex assaysSNP (Single Nucleotide Polymorphism) assay possibleHoweverMore expensive than DNA binding dyesMolecular BeaconsRQMolecularBeacon535353RQn n探针有核心的杂交区探针有核心的杂交区探针有核心的杂交区探针有核心的杂交区n n探针有自互补末端探针有自互补末端探针有自互补末端探针有自互补末端n n在未杂交时探针呈发夹状在未杂交时探针呈发夹状在未杂交时探针呈发夹状在未杂交时探针呈发夹状n n报道子报道子报道子报道子, , 荧光素在探针的荧光素在探针的荧光素在探针的荧光素在探针的 5 5 端端端端n n猝灭子在探针的猝灭子在探针的猝灭子在探针的猝灭子在探针的3 3 端端端端分子分子分子分子 BeaconsBeaconsn n当探针是发夹结构时，在报道子和猝灭子间有直当探针是发夹结构时，在报道子和猝灭子间有直当探针是发夹结构时，在报道子和猝灭子间有直当探针是发夹结构时，在报道子和猝灭子间有直接的能量转移接的能量转移接的能量转移接的能量转移Beacon Beacon 构造构造构造构造Molecular BeaconsAdvantages!Good for SNP (Single Nucleotide Polymorphism) detectionMultiplex assays possibleHoweverMolecular Beacons Are DIFFICULT to Design and SynthesizeDoes NOT generate a cumulative fluorescence signal, much weaker signal than the 5 Nuclease Assay (Cleavage probe)Expensive!A Few Words about ProbesHybridization oligos should have Tms 6 12 degrees higher than the associated primers.Avoid secondary structure in the complementary region of the probe.Avoid Gs at the 5 end of the probe sequence.Use oligo analysis tools to check probe for:DimerizationSecondary StructureCross Reactivity with Primers Each method has advantages and disadvantages Bio-Rad Real-Time Instrumentation is equipped to handle all chemistries One method may be more appropriate for an application over anotherWhich Method to Use?OutlineReal Time PCR and Conventional PCRIntroduction to Quantitative PCRCt value and Real-Time Quantitative PCR TheoryLaboratory TechniquePrimer Design for Real-Time PCRCt value and Real-Time Quantitative PCR Theory基线（基线（baseline) baseline) 阈值阈值(threshold)(threshold)基线（基线（baselinebaseline）的设置以）的设置以PCRPCR反应的前反应的前1515个循环的荧光信号作为荧光本底信号个循环的荧光信号作为荧光本底信号阈值阈值(threshold)(threshold)的设置一般是的设置一般是3-153-15个循个循环的荧光信号的标准偏差的环的荧光信号的标准偏差的1010倍。倍。PCRPCR扩增信号进入相对稳定对数增长期时扩增信号进入相对稳定对数增长期时的荧光值。的荧光值。阈值循环数阈值循环数CtCtPCRPCR扩增过程中荧光信号开始由本底进入指数增扩增过程中荧光信号开始由本底进入指数增长阶段所对应的循环次数，也就是荧光信号达长阶段所对应的循环次数，也就是荧光信号达到阈值时的循环次数。到阈值时的循环次数。从图中的重复实验中可以直观地看到，随着从图中的重复实验中可以直观地看到，随着PCRPCR反反应过程，荧光信号从基线经一个转折点进入指数应过程，荧光信号从基线经一个转折点进入指数期、线性期和最终的平台期，尽管平台期期、线性期和最终的平台期，尽管平台期DNADNA拷贝拷贝数波动很大，数波动很大，CTCT值却是相对固定的。值却是相对固定的。CtCt值与起始模板的关系值与起始模板的关系CycleThreshold Cycle, CTThe least?The least?Which one Which one has the has the most?most?如果用不同浓度模版如果用不同浓度模版DNADNA作作PCRPCR，可以看出模版，可以看出模版浓度越高，浓度越高，CTCT值越小。值越小。 模板起始浓度越高，Ct值越小 Ct值与模板起始拷贝数的对数存在线性关系Ct值与模板起始浓度的关系值与模板起始浓度的关系 如果模板浓度增加1倍，Ct值就提前1个循环到达 如果模板浓度减少1倍，Ct值就滞后1个循环到达1 CT Difference = 2 fold difference in starting template amount3.3 CT Difference = 10 fold difference in starting template amountProductT=(Template0)2nWhere n=Number of CyclesMathematic ImplicationsIdeal PCRThreshold Cycle, Ct, is a reliable indicator of initial copy number利用已知起始拷贝数的标准品可作出标准曲线，其中横坐标代表起始拷贝数的对数，纵坐标代表Ct值。因此，只要获得未知样品的Ct值，即可从标准曲线上计算出该样品的起始拷贝数。Absolute and Relative quantificationQuantificationNormalization of RT-PCR using reference genesDetermines changes in steady state transcription of a gene or genesExpression of the gene/s of interest is normalized against a reference gene/s (housekeeping gene/s) with no or insignificant expression variationExamples of some reference genes/housekeeping genes used : b-Actin, GAPDH, 18s rRNA b-2 Micro globulin, CyclophilinsBeta-Actin Ornithine decarboxylase (ODC)S-adenysyl methionine decarboxylase (SAMDC)Human Prostate and ThymusReal-Time Multiplex PCR: Gene ExpressionReal-time Multiplex Gene ExpressionBeta-ActinODCSAMDCProstate Ct : 23.25 Thymus Ct : 26.93Prostate Ct : 23.10 Thymus Ct : 25.53Prostate Ct : 21.25Thymus Ct : 21.90 Only possible with DNA Binding dyes (SYBR Green I) and after completed real time PCR Determines the temperature at which 50% of the DNA molecules separate into two strands - or “melts” apartDiscriminates by Melting Temperature (Tm), Tm is dependent on:- sequence (G/C content)- lengthWhat is Melt Curve Analysis?Fluorescence vs. Temperaturechange in fluorescencesingle amplified productTmMelt curve showing two amplified productsTwo amplified productsTmTmMelt CurveCheck specificity of the reactionCollecting at Higher Temperatures; does that work to avoid detection of primer dimers?Collecting at 82 C would record specific product only Primer Dimers Impact on the AssayOutlineReal Time PCR and Conventional PCRIntroduction to Quantitative PCRCt value and Real-Time Quantitative PCR TheoryLaboratory TechniquePrimer Design for Real-Time PCRLaboratory TechniqueGood Laboratory TechniqueDo not underestimate the importance of using:Screw cap tubesAerosol barrier tipsHot-start polymeraseMaster mixesReplicatesSerial dilutionsAnd the golden rule: Pipet only once into each wellPipet only once into each wellSame Reagents, Different HandsGood TechniquePoor TechniqueCycleCycleOutlineReal Time PCR and Conventional PCRIntroduction to Quantitative PCRCt value and Real-Time Quantitative PCR TheoryLaboratory TechniquePrimer Design for Real-Time PCRPrimer Design for Real-Time PCRPrimer Design for Real-Time PCRTargets an amplicon length of 75 to 200 bp50 to 60% overall GC contentLimit secondary structureLimit stretch of G or Cs longer than 3 basesNo stable interactions between primers (primer/dimer)Place Cs and Gs on ends of primers, but no more than 2 in the last 5 bases on 3 endPrimer Design:Step by Step1.Evaluate target sequence for secondary structure.2.Identify regions of 100 bp as potential amplicons.3.Scan sequence by eye or use a program to select potential primer pairs.*4.Use oligo analysis tools to check primers for:DimerizationSecondary StructureCross Reactivity *Repeat if necessary谢 谢 ！