An Introduction to Real-Time PCR

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1 An Introduction to Real-Time PCR 正茂生物科技有限公司產品專員童聖凱電話 : 信箱 : sk.tung@genmall.com.tw

2 Outline AMPLIFICATION Part I: What is the real-time PCR? Real-time PCR introduction Part II: How to use the real-time PCR? Operation and Data analysis

3 主要應用領域 AMPLIFICATION 基因表現藥物作用腫瘤指標基因調控基因治療微陣列基因晶片確認基因改造食品 (GMO) 病原菌偵測多重病原菌同時偵測 (Up to 5-Target) 適合大量檢體篩選 (High throughput) 定性, 定量同時完成藥物治療成效的監控 SNP 分型唯一可用 Taqman, Molecular Beacon 以及 FRET 三種方法的品牌

4 Conventional PCR vs QPCR +Taq +dntp +MgCl 2 +H 2 O +Buffer +Template +Primer (On ice) > 3 hrs(1.5 hrs labor) End point Fuzzy data Labor intensive +2x Master Mix +H 2 O +Template +Primer (Room Temp) Online monitoring More sensitive Automated <1 hrs (20 min labor)

5 中心法則 Central Dogma AMPLIFICATION Central dogma describes information flow from DNA RNA protein Protein considered the functional unit within the cell

6 Structure of DNA AMPLIFICATION

7 DNA Structure GC pair > AT pair

8 Classic PCR

9 Classic PCR

10 What is qpcr(real Time PCR)? Monitor the amplification reaction as it occurs End-point analysis of amplification product (30 cycles)

11 PCR: Theory vs. Reality AMPLIFICATION In theory, PCR reactions amplify exponentially, doubling every cycle and grow forever. In reality, Log Target DNA Theoretical increase Reality Cycle #

12 End Point Measurements 100 fold dilution series

13 Threshold Cycle, C T The point at which the fluorescence rises appreciably above background

14 Threshold Cycle (Ct) 與起始濃度的對數成反比 AMPLIFICATION 高濃度低濃度 Assumes 100% efficiency 1 cycle = 2 fold difference 3.3 cycles 10 fold difference

15 Absolute quantification T

16 Absolute quantification T

17 Absolute quantification 10 5 copies/well

18 C T is linear with the log of starting copy number The slope of the standard curve can be directly correlated to the efficiency of the reactions: Efficiency ( ) = [10 (-1/slope) ] - 1 when slope = -3.32, Efficiency = 100%

19 E Slope Slope = - [1/log (1+E)] log (1+E) = - (1/slope) E = 10 [-1/slope] -1 Aim for Efficiency Values: Good = % Fantastic = %

20 C T is linear with the log of starting copy number r = is a measure of how well the actual data fit to the standard curve. = (explained variation/total variation) Aim for R value (Correlation Coefficient) of 0.98

21 Points to Remember Threshold Cycle values (C T ) have a direct relationship to the amount of starting template 100% efficiency - 2 n = fold dilution Efficiency of reactions between % R value should be 0.98

22 Real-Time PCR These fluorescent molecules can be used Non-specific DNA binding dyes SYBR Green EvaGreen TM Specific Hybridization Probes/Primers TaqMan molecular beacons dual-oligo FRET pairs Scorpions /Amplifluor /LUX

23 SYBR Green I

24 DNA binding dyes Add Master Mix & Sample Primers 3 5 d. NTPs Thermal Stable DNA Polymerase 5 3 DNA binding dyes Reaction Tube 3 Denaturation Taq BD 5 Annealing 5 3

25 DNA binding dyes Extension 3 5 BD BD Taq 5 Taq BD BD BD Extension Continued Apply Excitation Wavelength 3 5 BD BD BD Taq 5 Taq BD BD 5 3 Repeat

26 DNA binding dyes AMPLIFICATION Easy to start qpcr Exp. Inexpensive Accurate Large dynamic range Primer specificity Single target

27 Melt Curve Analysis AMPLIFICATION After real-time PCR amplification, a melt curve is performed where we monitor fluorescence as temperature increases. Melting temperature (Tm) DNA is half double and half single-stranded Depends on nucleotide content (GC-AT ratio) and length Double Stranded DNA Single Stranded Tm

28 Melt Curve Analysis

29 Melt Curve Analysis AMPLIFICATION Distinguish products based on their Tms Plot negative rate of change of fluorescence vs. temp (-di/dt) for easy discrimination of products based on their Tms

30 Melt Curve Analysis

31 The DNA binding dye reagents of Bio-Rad Reagents iq SYBR Green Supermix Ssofast EvaGreen Supermix iscript One-Step RT-PCR Kit with SYBR Green Key Features Sensitive SYBR Green or EvaGreen Simplified reaction set up with ready-to-use 2x reaction mix itaq hot-start DNA polymerase, hot-start in 30 sec or 3 min at 95 Linear results over 7 orders of magnitude

32 Real-Time PCR These fluorescent molecules can be used Non-specific DNA binding dyes SYBR Green EvaGreen TM Specific Hybridization Probes/Primers TaqMan molecular beacons dual-oligo FRET pairs Scorpions /Amplifluor /LUX

33 Dyes and Quenchers FRET: Fluorescence (Förster) Resonance Energy Transfer F Q Dark Quenchers : Ground state or static quenching Energy is dissipated as heat Molecular interactions inhibit fluorescence F Q X

34 TaqMan the Hydrolysis probes

35 Cleavage-based assay: TaqMan Add Master Mix & Sample Primers 5 Reaction Tube R d. NTPs Thermal Stable DNA Polymerase Probe Q Denaturation Taq R 5 Q 5 3 Annealing

36 Cleavage-based assay: TaqMan Extension Step 3 Q Strand Displacement Taq 3 Q 5 3 R R R 5 2. Cleavage 3 Q Taq Taq 3. Polymerization Complete Detection Taq Q Q 5 3 R R 5

37 Cleavage-based assay: TaqMan More specific than dye base method Multiplex able using different reporter dye SNP genotyping application More high cost than dye base method

The probes based reagents of Bio-Rad Reagents iq Supermix Ssofast Probes Supermix iscript One-Step RT-PCR Kit for Probes iq Multiplex Powermix Key Features

38 The probes based reagents of Bio-Rad Reagents iq Supermix Ssofast Probes Supermix iscript One-Step RT-PCR Kit for Probes iq Multiplex Powermix Key Features Simplified reaction set up with ready-to-use 2x reaction mix hot-start DNA polymerase, hot-start in 30 sec or 3 min at 95 Linear results over 7 orders of magnitude

39 Which Method To Use? AMPLIFICATION 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 another -SNP, microarray, limited sample.

40 Real Time PCR application

41 Real Time PCR application AMPLIFICATION Quantitation Absolute viral load detection Relative Gene expression Identification Microorganisms Allelic discrimination, SNP

42 Types of quantification Absolute Determines input quantity of a nucleic acid target Requires a standard curve copies Normal Treatment A Treatment B Relative Determines fold differences in input target nucleic acid quantities between samples Performed with or without a standard curve Typically used for gene expression analysis with a target gene normalized to a reference gene

43 Absolute quantification

44 Absolute quantification T

45 Absolute quantification

46 Absolute quantification T

47 Absolute quantification 10 5 copies/well

48 Types of quantification Absolute Determines input quantity of a nucleic acid target Requires a standard curve copies Normal Treatment A Treatment B Relative Determines fold differences in input target nucleic acid quantities between samples Performed with or without a standard curve Typically used for gene expression analysis with a target gene normalized to a reference gene

49 Relative quantification Control C T = 21.3 Sample A C T = 22.8 Sample B C T = 19.3 T

50 Relative quantification Control C T = 21.3 = 1.5ng / tube 1 Sample A C T = 22.8 = 0.5ng / tube 0.33 Sample B C T = 19.3 = 6.0ng / tube 4 Fold

51 Gene Expression Normalization - Comparative C T Relative Quantity ( CT ) Not normalized Normalization accomplished via equal loading of samples Post analysis normalization Normalized Expression ( CT) Accounts for loading differences Usually normalize to reference gene Relative quantity of GOI is normalized by the relative quantity of the reference genes

52 Normalized Expression CT Assume 100% efficiency Simple Only one Ref Gene Pfaffl Modification Accounts for efficiency differences Only one Ref Gene Vandesompele Method Accounts for efficiency differences Allows multiple reference genes for normalization Complex

53 Relative Quantification - ΔCt GOI Tissue #1: 22 Tissue #2: 24 Delta Ct: = 2 Fold induction = 2 2 = 4

54 Comparative Ct Method (2-ΔΔCt) Tissue #1: Tissue #2: Reference GOI st Delta 2 nd Delta Delta Ct #1: = 1 Delta Ct #2: = 4 Delta Ct: 4-1 = 3 Fold induction = 2 3 = 8

55 Relative Quantification Problem with the CT C t % Starting quantity

56 Relative Quantification AMPLIFICATION Problem with the CT Slopes are not parallel C t % 100% Starting quantity

57 Relative Quantification Fold induction = deltact target (control-sample) Efficiency target deltact reference (control-sample) Efficiency reference (Pfaffl, 2001; Nucleic Acid Research) Efficiency = 10-1/slope

58 Relative Quantification Pfaffl Modification Tissue #1: Primer set #1Reference 21 Primer set #2 GOI 22 Tissue #2: (From Standard curve) Efficiency: 90% = % = 2 Delta Ct: = = 2 Fold induction = 2 target deltact target (24-22 = 2) 1.9 reference deltact reference (20-21 = -1) = 4 =

59 Methods Comparison AMPLIFICATION Ct method: ( no reference gene ) Fold induction : 4 Ct method: ( reference gene ) Fold induction : 8 Pfaffl modification: ( reference gene and efficiency ) Fold induction : 7.5

60 How to design Real Time PCR experiment

61 Gene Expression Workflow

62 Assay Considerations Gene structure Primer design Quality of RNA Reagent quality Pipetting Good lab practices

63 Amplicon Secondary Structures AMPLIFICATION

64 Effect of primer location AMPLIFICATION Primer A: = 66.3 % Primer A Primer B: = 95.8 % Reverse Primer B Primer B Forward Primer Reverse Primer A

65 Non validated primers 10,000 copies 2,000 copies 400 copies No Template

66 Non validated primers Same primers with a specific probe Poor resolution Poor replicates = 71%

67 Validated primers After primer re-design to eliminate primer-dimers r = = 91.3%

68 Fast Assay optimization dynamic thermal gradient

69 Fast Assay optimization dynamic thermal gradient 10 o C Above 6 o C Below

70 Gradient optimization Optimal annealing temperature

71 Fast Assay Optimization Test dynamic range

72 Sample and Template Preparation AMPLIFICATION Extract and analyze RNA Careful quantification is necessary RiboGreen Assay - Quantification Implen nanophotometer Quantification and purity Experion - Quality and quantification

73 Analysis of RNA purity and integrity AMPLIFICATION

74 Experion Analysis of RNA AMPLIFICATION Experiment: Evaluate sirna-mediated gene silencing Prevent faulty conclusions

75 Assay Considerations Reverse Transcription Efficiency Reproducible Data RNA Ideal Reality? cdna Not Reproducible

76 cdna Synthesis Serial diluted RNA & cdna iscript qrt-pcr Standard Curve Comparison: cdna serial dilution vs. total RNA serial dilution 40 T cdna Standard Total RNA Standard y e cdna total Slope Corr. Coef Intercept PCR efficiency 97.1% 97.6% d u Log Starting Quantity (femtograms of input RNA) Note: 1/10th of cdna reaction used for PCR

77 RNA to cdna AMPLIFICATION Prior to RT: Treat RNA with RNase-free DNase After RT: use enzyme that has RNaseH activity to digest away RNA from RNA:DNA hybrid

78 Assay Considerations Same Reagents, Different Hands Cycle Good Technique Cycle Poor Technique

79 Assay Considerations Improving Reproducibility Laboratory Techniques Use clean bench (hood) Use tips with filters Use calibrated micropipettors Use large volumes (3µL and up) Minimal pipetting into each reaction well

80 實驗配方 for iq SYBR green AMPLIFICATION 2X iq SYBR Green Supermix 12.5 ul Primer F (5 um) 2 ul Primer R (5 um) 2 ul ddh 2 O 3.5 ul Template 5 ul Total 25 ul

81 Conclusions Real-time PCR is a powerful technique that permits quantitative analyses to be performed Many detection strategies exist for qpcr, each requiring specific excitation and detection parameters Bio-Rad has the complete solution for all aspects of real-time PCR Gene Expression Gateway

82 Complete solutions AMPLIFICATION RQI value Random primers RNaseH Gradient function Multiple ref genes & efficiency

83 THANK YOU

Assay Design Considerations, Optimization and Validation

Assay Design Considerations, Optimization and Validation Ray Meng, Ph.D. International Field Applications Specialist Gene Expression Division Bio-Rad Laboratories, Inc. Assay Design Considerations Experiment