1、Bio-Rad定量PCR說明書,Bio-rad實(shí)時(shí)熒光定量PCR儀,BIO-RAD Gene Expression Division,Bio-Rad定量PCR說明書,Outline,Real Time PCR and Conventional PCR Introduction to Quantitative PCR Ct value and Real-Time Quantitative PCR Theory Laboratory Technique Primer Design for Real-Time PCR,Bio-Rad定量PCR說明書,Real Time PCR and Convent

2、ional PCR,Bio-Rad定量PCR說明書,技術(shù)背景,本來(lái)，PCR技術(shù)是為了將樣本中微量的DNA模版放大以便研究模版特性 隨著研究的深入，需要了解樣本中基因的表達(dá)模式與疾病的關(guān)系，這就要了解標(biāo)本中的DNA原始拷貝數(shù)。,Bio-Rad定量PCR說明書,Denaturation Primer Annealing Elongation,Repeat,常規(guī) PCR Process,In theory, product accumulation is proportional to 2n, where n is the number of amplification cycle repeats,

3、Bio-Rad定量PCR說明書,A linear increase follows exponential Eventually plateaus,Cycle #,Reality vs. Theory,Amplification is exponential, but the exponential increase is limited:,Bio-Rad定量PCR說明書,常規(guī)PCR方法的局限性分析：,無(wú)法對(duì)起始模板準(zhǔn)確定量,只能對(duì)終產(chǎn)物進(jìn)行分析 對(duì)終產(chǎn)物分析，受PCR過程平臺(tái)效應(yīng)的干擾，定量準(zhǔn)確度難以提高（相對(duì)誤差1000%）；存在擴(kuò)增產(chǎn)物的污染問題。 必須在擴(kuò)增后用電泳方法分析,費(fèi)時(shí)費(fèi)力而

4、且EB有毒 無(wú)法對(duì)擴(kuò)增反應(yīng)實(shí)時(shí)檢測(cè),Bio-Rad定量PCR說明書,C(t)s from 96 replicates are nearly identical, but the final fluorescence varies.,Cycle,End Point,PCR: Theory vs. Reality,實(shí)時(shí)定量PCR技術(shù)，是指在PCR反應(yīng)體系中加入熒光基團(tuán)，利用熒光信號(hào)積累實(shí)時(shí)監(jiān)測(cè)整個(gè)PCR進(jìn)程，使每一個(gè)循環(huán)變得“可見”，最后通過Ct值和標(biāo)準(zhǔn)曲線對(duì)樣品中的未知樣品 的起始濃度進(jìn)行定量的方法。,Real-Time PCR,Bio-Rad定量PCR說明書,Quantitative PCR

5、或 Real time PCR 是確定樣品中DNA (或 cDNA) 拷貝 數(shù)最敏感、最準(zhǔn)確的方法,Quantitative PCR /Real time PCR,Bio-Rad定量PCR說明書,C(t)s from 96 replicates are nearly identical, but the final fluorescence varies.,Cycle,End Point,C(t),PCR: Theory vs Reality,Bio-Rad定量PCR說明書,Quantitative information comes from monitoring the early sta

6、ges of amplification.,Cycle #,Theoretical,Real Life,Log Target DNA,Detector,定量的最佳時(shí)期,Bio-Rad定量PCR說明書,常用的定量方法,相對(duì)定量：相對(duì)于另一參照樣本的量； 絕對(duì)定量：用標(biāo)準(zhǔn)品作標(biāo)準(zhǔn)曲線來(lái)推算未知的樣本的量。,Bio-Rad定量PCR說明書,Quantitative PCR和常規(guī)PCR技術(shù)的區(qū)別,常規(guī)PCR是通過電泳對(duì)擴(kuò)增反應(yīng)的最終產(chǎn)物進(jìn)行定性分析（定量不準(zhǔn)確）； Quantitative PCR是在PCR反應(yīng)體系中加入熒光基團(tuán)，利用熒光信號(hào)積累實(shí)時(shí)監(jiān)測(cè)整個(gè)PCR進(jìn)程，使每一個(gè)循環(huán)變得“可見”，通過C

7、t值和標(biāo)準(zhǔn)曲線對(duì)樣品中的DNA (or cDNA) 的起始濃度進(jìn)行定量的方法（準(zhǔn)確定量） 。,Bio-Rad定量PCR說明書,Introduction to Quantitative PCR,PCR儀 + 監(jiān)測(cè)、分析系統(tǒng) + 熒光標(biāo)記,采用半導(dǎo)體加熱和制冷 加熱速度 3.3C /秒 ，制冷速度 2C/秒 升降溫速度可調(diào) 控溫準(zhǔn)確性：0.3,定量PCR儀應(yīng)該具備的特點(diǎn)：杰出的溫度控制,Bio-Rad定量PCR說明書,溫度梯度選擇可以允許使用者在同一次擴(kuò)增中優(yōu)化復(fù)性溫度。,定量PCR儀應(yīng)該具備的特點(diǎn)：梯度PCR功能,采用多點(diǎn)溫控和傳感技術(shù)，可以實(shí)現(xiàn)梯度PCR功能,采用0.2ml的離心管, 排管和

8、96孔PCR反應(yīng)板。 降低消耗品成本。,定量PCR儀應(yīng)該具備的特點(diǎn)： 消耗品成本低，樣品容量大,同時(shí)擴(kuò)增和檢測(cè)96個(gè)樣品,Bio-Rad定量PCR說明書,Introduction to Quantitative PCR,PCR儀 + 監(jiān)測(cè)、分析系統(tǒng) + 熒光標(biāo)記,Bio-Rad定量PCR說明書,定量PCR儀應(yīng)該具備的特點(diǎn)： 先進(jìn)的光路設(shè)計(jì)-多孔樣品同時(shí)檢測(cè),Bio-Rad定量PCR說明書,Fluorescence Detection,Bio-Rad定量PCR說明書,Multiplexing-同時(shí)檢測(cè)5色熒光,Bio-Rad定量PCR說明書,Cycle,Log Relative Fluoresc

9、ence,Multiplexing,Bio-Rad定量PCR說明書,定量PCR儀應(yīng)該具備的特點(diǎn)：開放性的系統(tǒng),可兼容的定量方法： SYBR Green I、Taqman探針、Beacon探針、FRET探針,可兼容的突變檢測(cè)方法： 熔點(diǎn)曲線功能、MGB探針,可兼容的試劑種類： 所有國(guó)產(chǎn)與進(jìn)口試劑,Bio-Rad定量PCR說明書,iQ5 Data Analysis Software,iQ5 Data Analysis Software,Bio-Rad定量PCR說明書,定量PCR儀應(yīng)該具備的特點(diǎn)： 分析軟件功能強(qiáng)大簡(jiǎn)潔直觀,Bio-Rad定量PCR說明書,異常數(shù)據(jù)或情況提示,Bio-Rad定量PCR

10、說明書,標(biāo)準(zhǔn)曲線 定量計(jì)算,自動(dòng)數(shù)據(jù)分析,Bio-Rad定量PCR說明書,多種報(bào)告單模式可供選擇-尤其重要,Bio-Rad定量PCR說明書,基因表達(dá)分析,Bio-Rad定量PCR說明書,Introduction to Quantitative PCR,PCR儀 + 監(jiān)測(cè)、分析系統(tǒng) + 熒光標(biāo)記,Bio-Rad定量PCR說明書,Detection Chemistries,Bio-Rad定量PCR說明書,Non-specific DNA binding dyes SYBR Green I SYBR Gold Ethidium Bromide Specific Hybridization Probe

11、s Cleavage Probes (TaqManTM) Molecular beacons ScorpionsTM AmplifluorTM LUXTM dual-oligo FRET pairs,Quantitative PCR Detection Chemistries,Bio-Rad定量PCR說明書,DNA Binding Dyes,5,Bio-Rad定量PCR說明書,Extension,Extension Continued Apply Excitation Wavelength,Repeat,DNA binding dyes,Bio-Rad定量PCR說明書,DNA Binding

12、Dyes,Advantages! Inexpensive compared to hybridization probes No 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 formation Multiplex assays not possible,Bio-Rad定量PCR說明書,SYBR Gr

13、een I Experiment,Typical “first step” experiment: Evaluate Primer Specificity Using Melt Curve Analysis Evaluate Primer Pair Efficiencies By running serial dilutions of template as standards Identify Sub-Optimal aspects of assay Optimize further with thermal gradient, etc.,Bio-Rad定量PCR說明書,Cleavage P

14、robes (TaqManTM),5,3,Q,F,Taq,5,q,3,l,3,5,Bio-Rad定量PCR說明書,雜交探針的互補(bǔ)區(qū)在引物間 5端連有一個(gè)熒光reporter, 通常是熒光素 3端連有一個(gè)Quencher,通常是TAMRA 熒光素被488 nm光激發(fā)，發(fā)射光為520 nm 520 nm 光能激發(fā)TAMRA，發(fā)射光為570 nm,TaqMan,Bio-Rad定量PCR說明書,Cleavage-based assay: TaqManTM,d.NTPs,Thermal Stable DNA Polymerase,Primers,Add Master Mix and Sample,Den

15、aturation,Annealing,Reaction Tube,l,Bio-Rad定量PCR說明書,5,3,Extension Step,1. Strand Displacement,2. Cleavage,3. Polymerization Complete,4. Detection,l,Cleavage-based assay: TaqManTM,Bio-Rad定量PCR說明書,Cleavage Probes (TaqManTM),Advantages! Generates a robust cumulative fluorescence signal Simple to design

16、 and synthesize compared to other hybridization probes (i.e. beacons) Ideal approach for multiplex assays SNP (Single Nucleotide Polymorphism) assay possible However More expensive than DNA binding dyes,Bio-Rad定量PCR說明書,Molecular Beacons,R,Q,Molecular Beacon,5,3,R,Q,Bio-Rad定量PCR說明書,探針有核心的雜交區(qū) 探針有自互補(bǔ)末端

17、 在未雜交時(shí)探針呈發(fā)夾狀 報(bào)道子, 熒光素在探針的 5 端 猝滅子在探針的3 端,分子 Beacons,Bio-Rad定量PCR說明書,當(dāng)探針是發(fā)夾結(jié)構(gòu)時(shí)，在報(bào)道子和猝滅子間有直接的能量轉(zhuǎn)移,Beacon 構(gòu)造,Bio-Rad定量PCR說明書,Molecular Beacons,Advantages! Good for SNP (Single Nucleotide Polymorphism) detection Multiplex assays possible However Molecular Beacons Are DIFFICULT to Design and Synthesize D

18、oes NOT generate a cumulative fluorescence signal, much weaker signal than the 5 Nuclease Assay (Cleavage probe) Expensive!,Bio-Rad定量PCR說明書,A Few Words about Probes,Hybridization oligos should have Tms 6 12 degrees higher than the associated primers. Avoid secondary structure in the complementary re

19、gion of the probe. Avoid Gs at the 5 end of the probe sequence. Use oligo analysis tools to check probe for: Dimerization Secondary Structure Cross Reactivity with Primers,Bio-Rad定量PCR說明書,Each method has advantages and disadvantages Bio-Rad Real-Time Instrumentation is equipped to handle all chemist

20、ries One method may be more appropriate for an application over another,Which Method to Use?,Bio-Rad定量PCR說明書,Outline,Real Time PCR and Conventional PCR Introduction to Quantitative PCR Ct value and Real-Time Quantitative PCR Theory Laboratory Technique Primer Design for Real-Time PCR,Bio-Rad定量PCR說明書

21、,Ct value and Real-Time Quantitative PCR Theory,Bio-Rad定量PCR說明書,基線（baseline) 閾值(threshold),基線（baseline）的設(shè)置以PCR反應(yīng)的前15個(gè)循環(huán)的熒光信號(hào)作為熒光本底信號(hào) 閾值(threshold)的設(shè)置一般是3-15個(gè)循環(huán)的熒光信號(hào)的標(biāo)準(zhǔn)偏差的10倍。 PCR擴(kuò)增信號(hào)進(jìn)入相對(duì)穩(wěn)定對(duì)數(shù)增長(zhǎng)期時(shí)的熒光值。,Bio-Rad定量PCR說明書,閾值循環(huán)數(shù)Ct,PCR擴(kuò)增過程中熒光信號(hào)開始由本底進(jìn)入指數(shù)增長(zhǎng)階段所對(duì)應(yīng)的循環(huán)次數(shù)，也就是熒光信號(hào)達(dá)到閾值時(shí)的循環(huán)次數(shù)。,Bio-Rad定量PCR說明書,從圖中的重復(fù)實(shí)

22、驗(yàn)中可以直觀地看到，隨著PCR反應(yīng)過程，熒光信號(hào)從基線經(jīng)一個(gè)轉(zhuǎn)折點(diǎn)進(jìn)入指數(shù)期、線性期和最終的平臺(tái)期，盡管平臺(tái)期DNA拷貝數(shù)波動(dòng)很大，CT值卻是相對(duì)固定的。,Ct值與起始模板的關(guān)系,Bio-Rad定量PCR說明書,Threshold Cycle, CT,如果用不同濃度模版DNA作PCR，可以看出模版濃度越高，CT值越小。,Bio-Rad定量PCR說明書,模板起始濃度越高，Ct值越小 Ct值與模板起始拷貝數(shù)的對(duì)數(shù)存在線性關(guān)系,Ct值與模板起始濃度的關(guān)系,如果模板濃度增加1倍，Ct值就提前1個(gè)循環(huán)到達(dá) 如果模板濃度減少1倍，Ct值就滯后1個(gè)循環(huán)到達(dá),Bio-Rad定量PCR說明書,1 CT Diff

23、erence = 2 fold difference in starting template amount 3.3 CT Difference = 10 fold difference in starting template amount,ProductT=(Template0)2n Where n=Number of Cycles,Mathematic Implications,Ideal PCR,Bio-Rad定量PCR說明書,Threshold Cycle, Ct, is a reliable indicator of initial copy number,利用已知起始拷貝數(shù)的標(biāo)準(zhǔn)

24、品可作出標(biāo)準(zhǔn)曲線，其中橫坐標(biāo)代表起始拷貝數(shù)的對(duì)數(shù)，縱坐標(biāo)代表Ct值。因此，只要獲得未知樣品的Ct值，即可從標(biāo)準(zhǔn)曲線上計(jì)算出該樣品的起始拷貝數(shù)。,Bio-Rad定量PCR說明書,Bio-Rad定量PCR說明書,Absolute and Relative quantification,Quantification,Bio-Rad定量PCR說明書,Normalization of RT-PCR using reference genes,Determines changes in steady state transcription of a gene or genes Expression of

25、the gene/s of interest is normalized against a reference gene/s (housekeeping gene/s) with no or insignificant expression variation Examples of some reference genes/housekeeping genes used : b-Actin, GAPDH, 18s rRNA b-2 Micro globulin, Cyclophilins,Bio-Rad定量PCR說明書,Beta-Actin Ornithine decarboxylase

26、(ODC) S-adenysyl methionine decarboxylase (SAMDC) Human Prostate and Thymus,Real-Time Multiplex PCR: Gene Expression,Bio-Rad定量PCR說明書,Real-time Multiplex Gene Expression,Beta-Actin,ODC,SAMDC,Prostate Ct : 23.25 Thymus Ct : 26.93,Prostate Ct : 23.10 Thymus Ct : 25.53,Prostate Ct : 21.25 Thymus Ct : 21

27、.90,Bio-Rad定量PCR說明書,Bio-Rad定量PCR說明書,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” apart Discriminates by Melting Temperature (Tm), Tm is dependent on: - sequence

28、 (G/C content) - length,What is Melt Curve Analysis?,Bio-Rad定量PCR說明書,Fluorescence vs. Temperature,Bio-Rad定量PCR說明書,Melt curve showing two amplified products,Melt Curve Check specificity of the reaction,Bio-Rad定量PCR說明書,Collecting at Higher Temperatures; does that work to avoid detection of primer dime

29、rs?,Collecting at 82 C would record specific product only,Primer Dimers Impact on the Assay,Bio-Rad定量PCR說明書,Outline,Real Time PCR and Conventional PCR Introduction to Quantitative PCR Ct value and Real-Time Quantitative PCR Theory Laboratory Technique Primer Design for Real-Time PCR,Bio-Rad定量PCR說明書,Laboratory Technique,Bio-Rad定量PCR說明書,Good Laboratory Technique,Do not underestimate the importance of using: Screw cap tubes Aerosol barrier tips Hot-start polymerase Master mixes Replicates Serial dilutions And the golden rule: Pipet only once into each well,Bio-Rad定量PCR說明書,Sam