Real Time PCR Basic Theory & Experiment Design. Summary Real Time Basic Theory Experiment Design & optimization.

Real Time PCR Basic Theory & Experiment Design

Summary Real Time Basic Theory Experiment Design & optimization

Denaturation Primer Annealing Elongation 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ Taq Repeat The PCR Process In theory, product accumulation is proportional to 2 n, where n is the number of amplification cycle repeats

Theoretical Real Life Log Target DNA n Exponential increase is limited n Linear increase follows exponential n Eventually plateaus Theory vs. Reality Real Time PCR utilizes fluorescence detection technology to allow us to monitor a reaction while it is occurring.

Quantitative information comes from monitoring the early stages of amplification Cycle # Theoretical Real Life Log Target DNA DetectorDetector How is quantitative data collected?

Real-Time PCR Process In real-time PCR reactions, fluorescent molecules are used to monitor the reaction while amplification is taking place.

Detection Chemistries ( 检测中的化学荧光染料 )

Real-Time PCR Detection These fluorescent molecules can be –Non-specific DNA binding dyes SYBR ® Green I –Specific Hybridization Probes/Primers TaqMan ™ Molecular Beacons Dual-oligo FRET pairs Scorpions ™ /Amplifluor ™ /LUX ™

DNA Binding Dyes u Intercalating Dyes are inexpensive compared to hybridization probes. u A dye based strategy allows one to get a general confirmation of amplification. u SYBR Green, is a more sensitive intercalating dye q SYBR Green I fluoresces 1000 times more brightly when bound to dsDNA

5’ 3’ d. NTPs Thermal Stable DNA Polymerase Primers 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Denaturation 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Annealing DNA Binding Dyes SYBR Green I Taq SG Add iQ SYBR™ Green Supermix, Primers & Sample

Extension 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Extension Continued Apply Excitation Wavelength 5’ 3’ 5’ 3’ 5’ Taq 3’ 5’ 3’ Taq 5’ Repeat DNA Binding Dyes SG

DNA Binding Dyes Advantages  Inexpensive compared to hybridization probes  No additional design work than the primer used for PCR reaction Disadvantages  Not template specific, will bind ALL double stranded DNA inducing primer-dimer and unspecific amplicon formation  Multiplex assays not possible

DNA Binding Dyes 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.

5’ 3’ d. NTPs Thermal Stable DNA Polymerase Primers 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Add iQ Supermix, Hybridization Probe and Sample Denaturation 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Annealing Taq 5’ 3’ R Q Probe 5’ 3’ R Q TaqMan®

5’ 3’ 5’ 3’ TaqMan® 5’ 3’ R Q 5’ 3’ Taq 3’ QR 5’ 3’ Q Taq R 5’ 3’ Q Taq R 3’ 5’ Primer Extension Cleavage Polymerization Detection 5’ 3’ Q Taq R 5’ R

TaqMan Probes Advantages Generates a robust cumulative fluorescence signal Simple to design and synthesize compared to other hybridization probes (i.e. beacons) Ideal approach for multiplex assays SNP (Single Nucleotide Polymorphism) assay possible Disadvantages More expensive than DNA binding dyes

Detection Chemistries 2% 3% 9% 15% 19% 78% 0%10%20%30%40%50%60%70%80% TaqMan probes Molecular Beacons FRET probes LUX fluorogenic primers MGB Eclipse probes Other Scorpion probes Commonly Used Fluorescent Probes

Each method has advantages and disadvantages Bio-Rad Real-Time Instrumentation is equipped to handle all chemistries (CFX 96 is optimized for FRET ) One method may be more appropriate for an application over another Which Chemistry To Use?

Dye/Quencher selection Select dyes with excitation/emission maxima compatible with the excitation/detection ranges of the instrument. Select the appropriate quencher for each dye Select non-fluorescent quenchers (e.g. BHQs, Dabcyl) instead of TAMRA Preferentially select dyes with good performance (usually indicated by high extinction coefficients and quantum yields) When multiplexing, strive for minimal spectral overlap between dyes Label your least abundant target with the best performing dye (usually FAM)

The point at which the fluorescence rises appreciably above background What is Threshold Cycle (CT)?

Threshold Cycle, C T Correlates strongly with the starting copy number Is linear with the log of starting copy number The least? Which one has the most?

Threshold Cycle, C T Of the same 96 replicates shows nearly identical values

End-point vs. Real-Time Lockey et al. (1998) Biotechniques 24:744-6

r = is a measure of how well the actual data fit to the standard curve. = (explained variation/total variation) The slope of the standard curve can be directly correlated to the efficiency of the reactions: Efficiency (  ) = [10 (-1/slope) ] - 1 Threshold Cycle, CT, is a reliable indicator of initial copy number

Standard Curve

10 8 10 6 10 2 10 4 T Real time PCR

10 5 copies/well Real time PCR

Real-Time Assay Options As with all real-time qPCR, analysis options include –Absolute Quantitation –Relative Quantitation Using Standard Curve Using Algorithm (e.g. 2 -  Ct ) Most gene expression analyses are interested in relative expression (i.e. comparing expression in one sample to another)

Relative Quantification Methods Δ Ct method: (no reference gene) Δ Δ Ct method: (reference gene,same efficiency) Pfaffl modification: (reference gene and efficiency) Vandesompele: (Multiple reference gene)

GOI Tissue #2: Tissue #1:22 24 Δ Ct:24-22 = 2 Fold induction =2 2 = 4 ΔCt

ReferenceGOI 21 Tissue #2: Tissue #1: 20 22 24 Δ Ct #2: Δ Ct #1:22-21 = 1 24-20 = 4 1 st Delta Δ Δ Ct:4-1 = 32 nd Delta Fold induction =2 3 = 8 (2-ΔΔCt)

CtCt Starting quantity 90% 24 22 Problem with the Δ Δ C T

CtCt 90% 24 22 100% Starting quantity

Relative Quantification E GOI (C T GOI (control) - C T GOI (sample) ) E REF (C T REF (control) - C T REF (sample) ) = Relative Expression (sample) GOI = Gene of Interest REF = Reference Gene

Primer set #1ReferencePrimer set #2 GOI 21 Tissue #2: Tissue #1: 20 22 24 90% = 1.9 Delta Ct: Efficiency: 20-21 = -1 100% = 2 24-22 = 2 2 target deltaCt target (24-22 = 2) Fold induction = 1.9 reference deltaCt reference (20-21 = -1) = 4 0.53 = 7.5 0.53 7.5 (From Standard curve) Pfaffl method

Δ Ct method: (no reference gene) Fold induction : 4 Δ Δ Ct method: (reference gene,same efficiency) Fold induction : 8 Pfaffl modification: (reference gene and efficiency) Fold induction : 7.5

Use of reference (normalizer) genes Used to control for differences between samples: –Amount of starting material (RNA isolation) –Efficiency of cDNA synthesis –Overall transcriptional activity of tissues or cells Normalizes target gene so that it is expressed as number of copies per copy of reference gene, rather than absolute copy number

An ideal reference gene Should not vary in expression in the tissues or cells under investigation Should not vary in expression in response to experimental treatment Must be validated for each assay

Commonly Used Reference Genes

Use of Multiple Reference Genes

Normalization Factor (NF)

About cDNA Synthesis Reverse transciption of mRNA to cDNA A number of priming options –Random oligos (e.g. random hexamers) Primes all RNA (not just mRNA); non-specific Can overestimate mRNA copy numbers Creates a cDNA pool for multiple, subsequent experiments Allows analysis of multiple targets –Oligo dTs Primes only mRNA; hybridizes to 3’ poly A tail Requires high quality, full length RNA More specificity than random oligos Creates a cDNA pool for multiple, subsequent experiments Allows analysis of multiple targets –Gene-specific primers Most specific option; primes only RNA for the gene of interest Highest yield of specific product Requires separate priming reaction for each target

Reverse Transcriptases MMLV (Moloney Murine Reverse Transcriptase): Lower activity temp; 37  C Lower intrinsic Rnase H activity Better for full length or longer cDNAs (making libraries) AMV (Avian Myoblastosis Virus): More robust than MMLV Higher intrinsic Rnase H activity Higher activity temp 41  C Eliminates problems with RNA secondary structure Tth (Thermus thermophilus): Both RT and DNA polymerase High activity temp, 68-74  C Significantly less efficient than either above

Saturating dye technology for HRM - LCGreen ™ I, EVA Green, Syto 9 LC Green ™ I Saturation dyes are less toxic, so concentration used can be high enough to allow all sites to be saturated Saturation eliminates potential for dye relocation-ideal for HRM SYBR ™ Green I is toxic to PCR, so concentration used is very low Intercalation Chemistries SYBR ® Green I Unsaturated binding allows dye to relocate as melting begins About HRM

HRM Profile 0.02deg

Data Acquisition Melting curves-normalized by selecting linear regions before and after the melting transition Two regions defined-upper 100% double stranded and lower single stranded baseline

Homoduplexes C or T Homozygotes represented by a single base change are differentiated by a difference in Tm melt. T AT A CGCG

Heteroduplex C>T Heterozygotes form heteroduplexes, the heterozygote (blue) trace is a mix of 4 duplexes CGCG TATA TGTG CACA + CGCG TATA + +

ACTN3 (R577X) (C—T). 10 replicates. 40 cycle fast (~34 min). SOFTWARE: Normalised HRM data

Real-Time PCR: Applications Real-Time reaction monitoring provides information for relative or quantitative measurements of starting material.  Gene Expression Studies  Microarray Validation  Transgenic Analysis  GMO Testing  Viral/Bacterial Load Studies  Molecular Diagnostics  Allelic Discrimination

Experimental Design How to Obtain Exceptional Real-Time PCR Results

What makes a good PCR reaction? Good Laboratory Practices Good Primer/Probe Design Good Amplicon Design High Quality Template Optimal Reagent Concentrations Good Instrument Performance Optimal Cycling Protocols Good Experimental Design – Controls, Replicates, Standards, Testing Assumptions

General Laboratory Practices Use clean bench (hood) Wear gloves Use screwcap tubes Use aerosol-resistant filter tips Use calibrated pipettes dedicated to PCR Use large volumes (>5  l) Use PCR-grade water Use a hot-start polymerase Use master mixes Pipette only once into each tube

Same Reagents, Different Hands Good Technique Poor Technique Cycle

Experiment Design Primer/probe design (Target/Reference) Validation by SYBR Green,for primer/probe efficiency / specificity/ reproducibility Standards preparation

Good Primer/Amplicon Design Maximizes reaction efficiency Maximizes specificity Maximizes yield/sensitivity Minimizes non-specific amplification Minimizes cross-reactivity in multiplex reactions Accurate, Reproducible Results

Real-Time PCR Primer Design Targets an amplicon length of 70 to 400bp (70~150bp for probe-based assays,100~400bp for SYBR Green) 30 to 80% overall GC content(ideally 50-60%) Maintain a melting temperature (Tm) between 50 and 65ºC  Tm (difference in Tm between FWD and REV primers) should be less than 2 o C

Real-Time PCR Primer Design Limit stretch of G ’s or C’s longer than 3 bases Limit secondary structure No stable interactions between primers (primer/dimer) Place C’s and G’s on ends of primers, but no more than 2 in the last 5 bases on 3’ end Always BLAST your primers (http://www.ncbi.nlm.nih.gov/blast/)

Real-Time PCR Probe Design Probe length should be between 18 and 30 bp (ideally 20 bp) GC content of probe should be 30-80% (ideally 40-60%) Tm of probe should be 10 o C higher than primers 3’ end of primer and 5’ end of probe on same strand between 1-10 bp distance Avoid secondary structure in the complementary region of the probe

Real-Time PCR Probe Design Avoid runs of > 3 identical nucleotides (especially G’s) within the probe Avoid G’s at the 5’ end of the probe sequence Use oligo analysis tools to check probe for: dimerization,secondary structure,cross reactivity with primers

Test primers with SYBR Green I –Efficiency By running serial dilutions of template as standards –Reproducibility By running replicates of some definite templates –Specificity Using Melt Curve Analysis Run agarose gel –Specificity To verify good primer/amplicon design…

Run SYBR Green assay using validated positive control for template (e.g. plasmid containing GOI) Include negative (no template) control Set up dilution series with 3-4 orders of magnitude Range of concentrations should include expected target concentration Run reactions in triplicate Run Melting Curve analysis To verify good primer/amplicon design…

Internet resource Real Time PCR Primer Sets http://www.realtimeprimers.org/ PrimerBank http://pga.mgh.harvard.edu/primerbank/index.html RT Primer DB http://medgen.ugent.be/rtprimerdb/ Quantitative PCR Primer Database (QPPD) http://lpgws.nci.nih.gov/cgi-bin/PrimerViewer Check for existing qPCR primers

Find Target Sequence Entrez Gene (NCBI) http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Gene Ensembl Genome Browser (Sanger Institute/European Bioinformatics Institute) http://www.ensembl.org/ Sequence Server (Dolan DNA Learning Center) http://www.dnalc.org/sequences/

Design Primers/Probes Primer 3 (Whitehead Institute, MIT) http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi GeneFisher (Bielefeld University) http://bibiserv.techfak.uni-bielefeld.de/cgi- bin/gf_submit?mode=STARTUP&qid=na&sample=dna http://bibiserv.techfak.uni-bielefeld.de/cgi- bin/gf_submit?mode=STARTUP&qid=na&sample=dna Fast PCR (Biocenter, University of Helsinki) http://www.biocenter.helsinki.fi/bi/bare-1_html/oligos.htm PerlPrimer (Owen Marshall) http://perlprimer.sourceforge.net/ Primer Design Assistant (Division of Biostatistics and Bioinformatics, National Health Research Institutes) http://dbb.nhri.org.tw/primer/

How to Prepare Serial Dilutions Stock Concentration of Plasmid = 10 8 copies/ml, 100ul Remove 10ul from 10 8 tube and add to the tube marked 10 7 Vortex and remove 10ul from 10 7 tube and add to the tube marked 10 6, etc. 10 8 10 5 10 6 10 7 10 4 10 3 10 2 10 1 Each tube contains 90  l H 2 0

Pipet enough template into one tube for the number of replicates you plan to run Add enough master mix to the replicate tube Vortex and spin before aliquoting into the experimental plate or tubes Pipet only once into wells or tubes How to Prepare Replicates

Check reaction efficiency, E, using standard curve Check reproducibility/linearity using R value Check specificity using Melting Curve Check for contamination using NTC Validation with SYBR Green

If efficiency is perfect (i.e amount of PCR product doubles each cycle), E=2 Calculate E using the slope of standard curve: E=10 -1/slope To calculate efficiency as a percentage: % Efficiency =(E - 1) x 100% Reaction Efficiency, E

E=10 -1/slope = 10 -1/-3.394 = 1.971 % Efficiency =(1.971 - 1) x 100% = 97.1%

Causes of Efficiencies <90% Poor primer/amplicon design –Non-specific amplification –Primer dimers –Secondary structure Non optimal cycling protocols –Annealing temperature too high (gradient optimization) –Extension time too short Non optimal reagent concentrations –Primers, polymerase, dNTPs, MgCl 2 PCR Inhibitors Inaccurate pipettes Poor laboratory procedures

 = 66.3 % Reverse primer A 1110 Maximizing Efficiency Primer Location

 = 95.8 % Reverse primer B Forward Primer 1 110 Reverse Primer B 2nd generation - 85 bp amplicon Maximizing Efficiency

Causes of Efficiencies >110% Poor primer design –Non-specific amplification Poor dilution series preparation Inaccurate pipettes Poor pipetting Dynamic range too large for limits of reaction

Gradient feature can be used to optimize primer and/or probe annealing conditions! Assay Optimization by Real-Time Gradient PCR

Reproducibility/Linearity The correlation coefficient, R (iQ software) are indicators of how well the data points fit the standard curve

R range from 0 to 1. If all data points lie perfectly on the line, value will be 1. R values are dependent on amount of variation and sample size. With 6 samples x 3 replicates: –R should be 0.992 or higher Reproducibility/Linearity

Specificity Single, well-defined peak in melting curve No amplification in negative control Verify specificity by running an agarose gel

DNA Template The source of template affects the accessibility of the target and must be considered during optimization It is important to optimize the reaction for the template concentrations that will be used in your experiment

DNA Template Genomic DNA (Intact, high MW DNA) –Cut with a restriction enzyme that does not cut region to be amplified –Boil DNA stock for 10 minutes and place immediately on ice Plasmid DNA –If there are problems with amplification, linearize the plasmid with a restriction enzyme that does not cut within the target cDNA –RNA must be free from genomic DNA contamination. Treat with RNAse-free DNase prior to reverse transcription. –Design primers at splice junctions to avoid genomic DNA amplification. –Ensure optimal, reproducible efficiency of RT reaction

Optimal Reagent Concentrations Primer Concentrations: –100~600nM(start with 300nM) –50~300nM(start with 150nM) for SYBR Green Probe Concentrations: –50~300nM(start with 200nM) Mg++ concentration: –3.5~5.5mM(start with 5mM) –1.5~3.5mM(start with 2.5mM) for SYBR Green

Controls Always include a positive control AND a negative control Controls, like samples, should be run in replicate Positive control –sample with predictable results –demonstrates that assay works –prevents “false negatives” from PCR inhibition (especially in +/- pathogen detection assays) Negative control –usually no template control –tests for contamination –if doing RT real-time PCR, include no RT controls to check for contamination by genomic DNAReplicates Real-time reactions should be run in replicate Ideally samples (and controls and standards) should be run in triplicate Acceptable variation between replicates depends on the mean and number of samples As a general rule, variation should be less than 0.5 Ct (ideally less than 0.25 Ct) To obtain good replicates: –Prepare a master mix with all reaction components, including the sample. –Use a hot start enzyme to prevent nonspecific amplification during preparation –Pipette once per well

Standards Appropriate template for standards includes : Plasmid containing gene of interest PCR product Synthetic oligo Positive control sample For Gene Expression assays: –cDNA from gene of interest –Genomic cDNA Standards should be quantified independently (e.g. UV Spectrophotometry, VersaFluor)

Standards A standard curve should include at least 5 different concentrations “Unknown” samples should fall within the limits of the standard curve; if they are outside the minimum/maximum standard concentration, quantification may not be accurate To quantify an unknown sample using a standard curve, the assumption is that the reaction efficiency of the standards is the same as the sample. It is NOT appropriate to import a standard curve from a different assay

Fluor selection 0 5000 0 0 Cy5 FAM ™ Texas Red ™ 490/530 575/620 635/680 Multiplexing

Testing primers Primers must amplify under the same conditions. Test primers with gradient concurrently to determine their actual annealing temperatures. Adjust annealing temp of primers by increasing and reducing their length. dynamic thermal gradient Multiplexing

Gene B Gene A Multiplexing

Not an optimal condition for multiplexing! Mix of two primer sets without template Multiplexing

Temperature Gradients Gene B Gene A

10 5 - 10 2 copies of GAPDH alone 10 5 - 10 2 copies of GAPDH with 10 9 copies of a -tubulin Concentration Differences

10 5 - 10 2 copies GAPDH alone 10 5 - 10 2 copies GAPDH with 10 9 copies of a-tubulin GAPDH primers = 250 nM a-tubulin primers = 25 nM0 Limiting Primers

2X enzyme, 2X dNTP, & Mg +2 Standard 1X conditions 3X enzyme, 2X dNTP, & Mg +2 r = 0.999 slope = -3.361 h=98% r = 0.979 slope = -4.542 h=66% Additional Reagents

~ Equal Efficiencies

Verifying Multiplex Reactions: Singleplex vs. Fourplex Cycle β-actin ODC OAZ 17.0 ± 0.0 17.3 ± 0.1 23.0 ± 0.2 23.1 ± 0.2 20.8 ± 0.1 20.8 ± 0.2 22.2 ± 0.1 AZI Multiplexing

Real Time PCR Basic Theory & Experiment Design. Summary Real Time Basic Theory Experiment Design & optimization.

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Real Time PCR Basic Theory & Experiment Design. Summary Real Time Basic Theory Experiment Design & optimization.

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