Q-PCR theory and points for attention

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Central Dogma Q-PCR theory and points for attention Central

DNA Structure Polymerase Chain Reaction Targeted The polymerase

thermostable 95DNA o targeting C DNA complementary polymerase of

complementary 50-65 to opposite o C strands of target region but

STRANDS PRIMERS 72 o C 72 o C Each cycle of PCR doubles the

as well) 1 cycle = 2 1 copies of starting template 50 65 o C 25

products No relationship between end point and starting target

molecules are used to monitor the reaction while amplification

1 Central Dogma Q-PCR theory and points for attention Central dogma describes information flow from DNA RNA protein 產品專員 / 陳立德 DNA Structure Polymerase Chain Reaction Targeted The polymerase use DNA of replication two primers synthesizes using allows thermostable 95DNA o targeting C DNA complementary polymerase of specific to sequences template in 5` to 3` direction Primers are complementary to opposite o C strands of target region but not complementary to any other sequences EXTEND ANNEAL DENATURE STRANDS PRIMERS 72 o C 72 o C Each cycle of PCR doubles the number of progeny DNA duplexes (which can then act as template as well) 1 cycle = 2 1 copies of starting template o C 25 cycles = copies o C of starting template PCR 1 st Generation What is Real-Time PCR? Amplify target DNA with end point analysis to distinguish products No relationship between end point and starting target copies Template Cycle 1 End point analysis Simply - fluorescent molecules are used to monitor the reaction while amplification is taking place. 2X Template Cycle 2 4X Template You are able to view this occurring in real-time on your instrument cycles 1

increase follows exponential 2) Eventually plateaus Real-Time PCR allows us to see the exponential phase so we can calculate how much we started with.

appreciably above background 96 replicates

2 Reality vs. Theory Quantitative PCR (qpcr) Amplification is exponential, but the exponential increase is limited: Real-time qpcr enables assessment of the reaction after each cycle 1) A linear increase follows exponential 2) Eventually plateaus Real-Time PCR allows us to see the exponential phase so we can calculate how much we started with. Log Target DNA Theoretical Cycle # Real Life t Product Amount 100 Conventional PCR 0 Cycle number 35 Threshold Cycle, C T End Point Measurements The point at which the fluorescence rises appreciably above background 96 replicates of an identical reaction can have very different final amounts of fluorescence Threshold Cycle, C t C T value v.s. concentration Correlates strongly with the starting copy number If you have twice the template, you get to C t one cycle earlier If you have half the template, you reach C t one cycle later Is linear with the log of starting copy number over six or more orders Human genomic DNA with SYBR 1 cycle = 2 fold difference 3.32 cycles 10 fold difference Assumes 100% efficiency Y= N0 2 n Y=N 0 (1+E) n ng of template- FV Leiden primers 2

3 Quantitative PCR (qpcr) C T is linear with the log of starting copy number (standard curve) Create a standard curve with 10-fold serial dilutions of PCR product assign arbitrary values Compare values from standards with values for unknown sample 100 Product Amount t STD 1: 1,000,000 STD 2: 100,000 STD 3: 10,000 STD 4: 1,000 STD 5: 100 STD 6: 10 Sample: 6,592 0 Cycle number 35 r = is The a measure slope of of the how standard well the curve actual can be data directly fit to correlated the standard to the curve. = (explained efficiency variation/total of the reactions: variation) Efficiency (E) = [10 (-1/slope) ] - 1 Aim for when R value slope (Correlation = -3.32, Efficiency Coefficient) = 100% of 0.98 E Slope Slope = - [1/log (1+E)] log (1+E) = - (1/slope) E = 10 [-1/slope] -1 Aim for Efficiency Values: Good = % Fantastic = % What are the most common detection strategies used for Real-Time PCR? Real-Time PCR DNA binding dyes These fluorescent molecules can be used Non-specific DNA binding dyes SYBR Green I Ethidium Bromide Add Master Mix & Sample d. NTPs Primers Thermal Stable DNA Polymerase DNA binding dyes Reaction Tube Specific Hybridization Probes/Primers Man molecular beacons dual-oligo FRET pairs Denaturation l BD Scorpions /Amplifluor /LUX Annealing 3

DNA binding dyes Melt Curve Analysis 3 5 3 5 Fluorescence decrease as the temperature increase: Extension 1. DNA strands start to separate 2.

Fluorescence rapidly decreases BD BD BD 5 3 Extension Continued Apply Excitation Wavelength l l l BD BD BD 3 5 BD BD 3 l l Repeat Melt Curve Analysis DNA

-Contaminating DNA, primer dimer or false priming is seen as an additional peak. Easy to start qpcr Exp.

4 DNA binding dyes Melt Curve Analysis Fluorescence decrease as the temperature increase: Extension 1. DNA strands start to separate 2. SYBR green looses its binding to the DNA 3 BD BD 5 3. Fluorescence rapidly decreases BD BD BD 5 3 Extension Continued Apply Excitation Wavelength l l l BD BD BD 3 5 BD BD 3 l l Repeat Melt Curve Analysis DNA binding dyes -The melting temperature of the amplicon can easily be detected. -Contaminating DNA, primer dimer or false priming is seen as an additional peak. Easy to start qpcr Exp. Inexpensive Accurate Large dynamic range Primer specificity Single target Cleavage-based assay: Man Probe Cleavage-based assay: Man Probe Q R Add Master Mix & Sample Primers d. NTPs Thermal Stable DNA Polymerase Extension Step 5 3 Reaction Tube R Probe Q 1. Strand Displacement Q 2. Cleavage 5 3 R Q R Denaturation Annealing l R Q 3. Polymerization 5 Complete 5 3 l 4. Detection R R 4

Probe-based assay More specific than dye base method Multiplex

More high cost than dye base method What data do you perform

Laborious techniques were used to examine specific gene

Northern I don t Blots see this Southern as 10 Blots fold.

to me! PCR Assay development was much easier.

Results were achieved much faster. Data sets grew larger.

Instruments generated numerical outputs.

5 Probe-based assay More specific than dye base method Multiplex able using different reporter dye SNP genotyping application More high cost than dye base method What data do you perform now? Historically before qpcr After the arrival of qpcr Laborious techniques were used to examine specific gene expression levels; 10 fold! Northern I don t Blots see this Southern as 10 Blots fold. It looks RNAse more Protection like 3 Assays Competitive fold to me! PCR Assay development was much easier. Sample required was much smaller. Results were achieved much faster. Data sets grew larger. Etc etc These methods were Simiquantitative at best. Instruments generated numerical outputs. The Problem QPCR papers Perceived simplicity of running qpcr experiments has resulted in many publications containing seriously misleading results. Key word search with Real-time PCR in NCBI Pubmed 5

6 Questionable Data Retracted Paper Slope : Eff = % Slope : Eff = % Autism and MMR Vaccine Autism and MMR Vaccine Lingering Effects of Bad Data What can be done? 6

MIBBI,RDML and MIQE A new beginning Promoting coherent minimum reporting guidelines for biological and biomedical investigations: the MIBBI project.

Lefever S, Hellemans J, Pattyn F, Przybylski DR, Taylor C, Geurts R, Untergasser A, Vandesompele J; on behalf of the RDML consortium. Nucleic Acids Res.

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. Clin Chem.

57 Essential information (E) must be submitted with the manuscript 28 Desirable information (D) should be submitted if available What is MIQE?

7 MIBBI,RDML and MIQE A new beginning Promoting coherent minimum reporting guidelines for biological and biomedical investigations: the MIBBI project. Nature biotechnology, volume 26, number 8, August 2008, RDML: structured language and reporting guidelines for real-time quantitative PCR data. Lefever S, Hellemans J, Pattyn F, Przybylski DR, Taylor C, Geurts R, Untergasser A, Vandesompele J; on behalf of the RDML consortium. Nucleic Acids Res Feb 17. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. Clin Chem Feb Essential information (E) must be submitted with the manuscript 28 Desirable information (D) should be submitted if available What is MIQE? It s a Checklist Experimental Design qpcr community driven guidelines for essential and desired information in litterature; Experimental Design Sample Information Nucleic Acid Extraction Reverse Transcription qpcr Target Information qpcr Oligonucleotides qpcr Protocol qpcr Validation Data Analysis Experimental Replicates Sample Information NTC 7

8 Sample Information Acquisition Sample Information Processing and Storage Source (soil, blood, urine, plant tissues ) Sampling techniques (biopsy materials, single-cell sampling, laser microdissection ) Description (percentage of the biopsy made up of tumor cells) Mass/Volume Homogeneity Processing procedure Storage (-80C vs -20C; duration ) Prep time to extraction (-80C) Sample degradationd Nucleic Acid Extraction Sample Extraction DNA / RNA Aquapure TM Genomic DNA Isolation Kit Aurum TM Total RNA Kits Contaminants Starches Lipids Metals Extraction contaminants Phenol chloroform Salts Sample degradation Inhibitors copurified 8

Effects of EDTA Template DNA Preparation Genomic DNA Cut with restriction enzyme that does not cut within amplicon Boil DNA for 10 min and then

with RNase-free DNase After RT: use enzyme that has RNaseH activity to digest away RNA from RNA:DNA hybrid Sample and Template Preparation

Quantification and purity Experion - Quality and quantification Experion Virtual Gel L C 3 5 10 15 1h 2h 4h Samples 6,7,8 are highly degraded.

44 3 min @ 90C 114 1.93 2.40 5 min @ 90C 115 2.06 2.37 10 min @ 90C 115 2.03 2.37 15 min @ 90C 116 2.02 2.31 1 hr @ 90C 109 1.99 2.

9 Effects of EDTA Template DNA Preparation Genomic DNA Cut with restriction enzyme that does not cut within amplicon Boil DNA for 10 min and then onto ice Plasmid DNA If it doesn t work, linearize plasmid with restriction enzyme that does not cut within amplicon cdna 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 Sample and Template Preparation Analysis of RNA purity and integrity Extract and analyze RNA Careful quantification is necessary RiboGreen Assay - Quantification NanoDrop Quantification and purity Experion - Quality and quantification Experion Virtual Gel L C h 2h 4h Samples 6,7,8 are highly degraded. RQI Value & Color Coded Classification Corresponding Nanodrop Readings Sample Conc (ng/ul) A260/280* A260/230* Control-no heat C C C C C C C *Generally accepted ratios (A260/280 and A260/230) for good quality RNA are > 1.8. Total RNA analysis Cq of 5 and 3 transcripts 9

Reverse Transcription RT protocols: Reverse transcription Strategies RNase is STRONG Oligo dt Plasmid extracted with traditional 3-steps and eluted with sterile ddh 2 O.

10 Reverse Transcription RT protocols: Reverse transcription Strategies RNase is STRONG Oligo dt Plasmid extracted with traditional 3-steps and eluted with sterile ddh 2 O. Random primers Oligo dt and Random primers Gene specific primers RNase H RNase inhibitors NO RNase Contain RNase Obtain Accurate Results potent blend of RNase inhibitor Increase primer design flexibility & prevent 5 3 bias optimum blend of oligo(dt) & random primers 5 3 AAAAA Notice the Ct delay RT with iscript (green trace) RT with iscript + RNaseA (red trace) RT with iscript without RNase inhibitor + RNase (black trace) RT 100, 10, 1, and 0.1 ng input RNA Primer pairs for 5 end (green trace - ~60 bp) Primer pairs for 3 end (orange trace - ~70 bp) 10

iscript works with a broad range of RNA Range

Competitors cannot reliably achieve this

and high amounts of input RNA g, 10-6 ng, 10-9

Bio-Rad s iscript cdna Synthesis supermix ~45

First-Strand Synthesis System for RT-PCR 13

qpcr Target Information NCBI Free resource

Amplicon Secondary Structures http://bioinfo.

11 iscript works with a broad range of RNA Range of iscript is100fg 1 g of input RNA Competitors cannot reliably achieve this range, and sometimes require two RTs for low and high amounts of input RNA g, 10-6 ng, 10-9 pg, fg, iscript Competitors Bio-Rad s iscript cdna Synthesis supermix ~45 min Completed Invitrogen s SuperScriptII First-Strand Synthesis System for RT-PCR 13 tubes! qpcr Target Information NCBI Free resource Blast sequence Stack sequence Blast analysis Amplicon Secondary Structures Bad location for primers Good location for primers h.gov/blast/blast.cgi 11

12 Effect of primer location qpcr Oligonucleotides Primer A: = 66.3 % Primer B: = 95.8 % Primer A Reverse Primer B Primer B Forward Primer Reverse Primer A Primer Design RTPrimerDB Designs Primers Designs Internal Oligos Provides multiple outputs Free Web software provided by Steve Rozen and Whitehead Institute for Biomedical Research. qpcr Protocol 12

Recipe, Protocol, Consumable, and Instrument The DNA binding dye reagents of Bio- Rad The probes based reagents of Bio-Rad Reagents iq SYBR Green Supermix iscript One-Step

Linear results over 7 orders of magnitude Reagents i universal supermix for Probe iscript One-Step RT-PCR Kit for Probes iq Multiplex Powermix Key Features Simplified

Reagents 3rd Generation Reagents: SsoFast TM Series Supermix SsoFast EvaGreen Supermix Sso7d from Sulfolobus solfataricus 7kD, 63 aa.

13 Recipe, Protocol, Consumable, and Instrument The DNA binding dye reagents of Bio- Rad The probes based reagents of Bio-Rad Reagents iq SYBR Green Supermix iscript One-Step RT-PCR Kit with SYBR Green Key Features Sensitive SYBR Green Simplified reaction set up with ready-to-use 2x reaction mix i hot-start DNA polymerase, hot-start in 3 min at 95 Linear results over 7 orders of magnitude Reagents i universal supermix for Probe 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 3rd Generation Reagents 3rd Generation Reagents: SsoFast TM Series Supermix SsoFast EvaGreen Supermix Sso7d from Sulfolobus solfataricus 7kD, 63 aa. Thermostable (Tm >90 C) No sequence preference Binds to dsdna (3-6 bp/protein molecule) Monomeric High Speed High Dynamic Range High Tolerance Minimal inhibition of PCR by use of EvaGreen Higher activity Tolerant to PCR inhibitors 13

Sso7d-fusion enzymes Enhanced processivity PCR Inhibition caused by Dye Enzyme Sso7d- Pfu Pfu-Sso7d Sso7d- Processivity 15-19nt 96-109nt 2-3nt 35-39nt 2-4nt 26-39nt

5kb 200bp 1.2kb 2.5kb Source: Barrett et al., Quantace Ltd.

Supermix ABI Fast SYBR Green Mastermix Fast Protocol 36 min <1.56% Efficiency = 104.0%, r = 0.992 Efficiency = 110.4%, r = 0.

14 Sso7d-fusion enzymes Enhanced processivity PCR Inhibition caused by Dye Enzyme Sso7d- Pfu Pfu-Sso7d Sso7d- Processivity 15-19nt nt 2-3nt 35-39nt 2-4nt 26-39nt 1x 0.75x 0.5x 0.25x 1.5x 2x 5x x Processivity is enhanced 5 to 10-fold Source: Barrett et al., Quantace Ltd. Even Dye Redistribution EvaGreen 1.2kb 200bp 2.5kb 200bp 1.2kb 2.5kb Source: Barrett et al., Quantace Ltd. The even dye redistribution gives equal signal intensities from different sized DNA fragments High Dynamic Range Phenol Chloroform (extraction mix) SsoFast EvaGreen Supermix ABI Fast SYBR Green Mastermix Fast Protocol 36 min <1.56% Efficiency = 104.0%, r = Efficiency = 110.4%, r = Invitrogen EXPRESS SYBR GreenER Qiagen QuantiFast SYBR Mix 3.125% Fast Protocol 36 min CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98 o C 30sec / 50x95 o C 1 sec 60 o C 5 sec / melt analysis Efficiency = 89.3%, r = Efficiency = 98.5%, r = Bio-Rad Mix in Green Competitor Mix in Blue 14

Phenol Chloroform (extraction mix) Blood Serum <0.78% 1.56% <0.39% 0.

95 o C 5min / 50x 95 o C 15 sec 60 o C 60 sec / melt analysis <2.5 5% 10 % <0.78% 1.

56% CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98 o C 30sec / 50x95 o C 1 sec 60 o C 5 sec / melt analysis CCl26 amplified using Other Reagent B:

melt analysis Blood Serum Inhibitor Concentration Not Having Any Significant Effect on qpcr Reaction <0.0098 % 0.039 % <0.0089% 0.

18 % 4.375 % 4.375 % KCl 500 mm 2 fold 62.5 mm 31.25 mm 125 mm 62.5 mm 31.25 mm NaAc 250 mm 2 fold 62.5 mm 62.5 mm 62.5 mm 62.5 mm 15.

C 5min / 50x 95 o C 15 sec 60 o C 60 sec / melt analysis Phenol/Chloroform 6.25 % 2 fold 1.53 % 0.781 % 0.781 % 0.781 % 0.391 % Blood Serum 10 % 4 fold 2.5 % 0.0098 % 0.

15 Phenol Chloroform (extraction mix) Blood Serum <0.78% 1.56% <0.39% 0.78% CCl26 amplified using Bio-Rad iq SYBR Green Supermix: 5ul Assay95 o C 3 min / 50x 95 o C 10 sec 60 o C 60 sec / melt CCl26 amplified using Other Reagent A: 5ul Assay 95 o C 5min / 50x 95 o C 15 sec 60 o C 60 sec / melt analysis <2.5 5% 10 % <0.78% 1.56% <0.78% 1.56% CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98 o C 30sec / 50x95 o C 1 sec 60 o C 5 sec / melt analysis CCl26 amplified using Other Reagent B: 5ul Assay 95 o C 20sec / 50x 95 o C 3 sec 60 o C 30 sec / melt analysis CCl26 amplified using Other Reagent C: 5ul Assay 95 o C 20sec / 50x 95 o C 3 sec 60 o C 30 sec / melt analysis Blood Serum Inhibitor Concentration Not Having Any Significant Effect on qpcr Reaction < % % <0.0089% 0.039% Inhibitor Max Tested DilutionSer EVA iq SYBR ABI INV Euro ies Ethanol 25 % 2 fold 12.5 % 6.25 % % % % Isopropanol 17.5 % 2 fold 8.75 % % 2.18 % % % KCl 500 mm 2 fold 62.5 mm mm 125 mm 62.5 mm mm NaAc 250 mm 2 fold 62.5 mm 62.5 mm 62.5 mm 62.5 mm mm CCl26 amplified using Bio-Rad iq SYBR Green Supermix: 5ul Assay 95oC 3 min / 50x 95oC 10 sec 60oC 60 sec / melt CCl26 amplified using Other Reagent A: 5ul Assay 95 o C 5min / 50x 95 o C 15 sec 60 o C 60 sec / melt analysis Phenol/Chloroform 6.25 % 2 fold 1.53 % % % % % Blood Serum 10 % 4 fold 2.5 % % % % % <0.0089% 0.039% <0.0089% 0.039% Total Blood (+hep) 10 % 4 fold % % % % % Heparin U 2 fold U U U U U Soil 25 % 4 fold 6.25 % 1.56 % 1.56 % 6.25 % 0.39 % Coffee 25 % 4 fold 6.25 % 1.56 % 1.56 % 1.56 % 1.56 % CCl26 amplified using Other Reagent B: 5ul Assay 95 o C 20sec / 50x 95 o C 3 sec 60 o C 30 sec / melt analysis CCl26 amplified using Other Reagent C: 5ul Assay 95 o C 20sec / 50x 95 o C 3 sec 60 o C 30 sec / melt analysis qpcr Validation Optimization How to choose the annealing temperature? e 15

Evidence of optimization - thermal gradient Gradient optimization dynamic thermal gradient 10 o C

Curve Thermal gradient Dilution series 10 7 10 6 10 5 10 4 10 3 10 2 6 o C Below Gradient

dilution of template to test primers across a broad dynamic range.

highest reaction efficiency 62 o C Efficiency = 99% Efficiency = 98% Include representative unknown

16 Evidence of optimization - thermal gradient Gradient optimization dynamic thermal gradient 10 o C Above Single Plate Optimisation of Annealing Temperature and PCR Efficiency via Slope of Calibration Curve Thermal gradient Dilution series o C Below Gradient Optimization of Annealing Temperature SYBR Green Validation 67 o C Efficiency = 68% Use a serial dilution of template to test primers across a broad dynamic range. Serial dilutions at 8 temps from 55 o C to 68 o C Reactions at 62 o C annealing have low Cts and highest reaction efficiency 62 o C Efficiency = 99% Efficiency = 98% Include representative unknown samples. Evaluate specificity, efficiency, reproducibility and dynamic range. Gel checking. 56 o C Data Analysis qpcr Program/Software iq5 Optical System Software 16

Absolute Quantization Relative Quantization Gene Expression Normalization - Comparative C T Relative Quantification - Ct Relative Quantity (

Sample : 22 Control : 24 Delta etact: 22-24 = - 2 Accounts for loading differences Usually normalize to reference gene Relative quantity of

Expression Normalization - Normalized Expression Normalize Ct of target gene to that of reference gene Normalize ΔCt of sample to ΔCt of

24-20 = 4 Delta Ct: 1-4 = - 3 Fold induction = 2 -(-3) = 8 CT Assume 100% efficiency Only one Ref Gene Pfaffl Modification Accounts for

17 Absolute Quantization Relative Quantization Gene Expression Normalization - Comparative C T Relative Quantification - Ct Relative Quantity ( CT ) Not normalized Normalization accomplished via equal loading of samples Post analysis normalization Normalized Expression ( CT) GOI Sample : 22 Control : 24 Delta etact: = - 2 Accounts for loading differences Usually normalize to reference gene Relative quantity of GOI is normalized by the relative quantity of the reference genes Fold induction = 2 -(-2) = 4 Comparative Ct Method (2 - Ct ) Gene Expression Normalization - Normalized Expression Normalize Ct of target gene to that of reference gene Normalize ΔCt of sample to ΔCt of control sample 1 st Delta Reference GOI Tissue #1: (sample) Tissue #2: (control) nd Delta Delta Ct #1: = 1 Delta Ct #2: = 4 Delta Ct: 1-4 = - 3 Fold induction = 2 -(-3) = 8 CT Assume 100% efficiency 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 Simple Complex 17

Allelic Discrimination The RDML International Consortium Key developer group, a member community and

technote 5859 provides an excellent summary of a MIQE compliant experiment in practice, from start to

Profiling & Quantification Complete solutions RNA Analysis (Quality and Yield) Experion System

18 Allelic Discrimination The RDML International Consortium Key developer group, a member community and supporters 41 supporters and members from 20 different countries 22 academic Austria, Switzerland, France Spain, Israel, Canada,... Sweden (2) Germany (3) 19 companies USA (6) UK (2) Belgium (2) UK (3) USA (5) Portugal, Austria Switzerland, Israel, Australia, Korea,... Goal: develop and maintain the RDML data exchange format Conforming to MIQE: an example The Bio-Rad technote 5859 provides an excellent summary of a MIQE compliant experiment in practice, from start to finish Gene Modulation RNA Purification Aurum Total RNA Kits silentfect Transfectin Gene Pulser Xcell Profiling & Quantification Complete solutions RNA Analysis (Quality and Yield) Experion System SmartSpec Plus iscript cdna kits iq SYBR iscript Green Supermix reagents T100 Thermal SsoFast Cycler reagents CFX Series RQI value Reverse Transcription and Amplification Gradient function Multiple ref genes &efficiency qbase software Beacon Designer Questions? 18

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Introduction to Real-Time PCR: Basic Principles and Chemistries

Introduction to Real-Time PCR: Basic Principles and Chemistries Leta Steffen, PhD Applications Scientist Promega Corporation Outline I. Real-Time PCR overview Basics of Real-Time PCR Understanding the