Introduction to Real-Time PCR:

Basic Principles and Chemistries

Leta Steffen, PhD
Applications Scientist

Promega Corporation
©2013 Promega Corporation.
Outline

I. Real-Time PCR overview

• Basics of Real-Time PCR
• Understanding the data

II. Chemistries and Instrumentation

• Dye-based chemistries (e.g. SYBR)
• Label-based chemistries (e.g. TaqMan)
• Reverse Transcription qPCR
• Instrumentation

III. Quantification – an intro to the math

IV. The MIQE Guidelines

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PCR Refresher

Taq
Stage Temp Time Cycles
dNTPs
MgCl2 Activation 95°C 2 min 1
Buffer Dissociation 95°C 15 s 25 - 40 Gel electrophoresis
Primers Annealing 60°C 15 s
Template
Extension 72°C 1 min
Gel electrophoresis

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PCR Refresher

Denature Anneal Extend

~95°C ~55-65°C ~72°C

Start # Cycle1 Cycle2 Cycle3 Cycle4 Cycle5 Cycle6 Cycle n

1 2 4 8 16 32 64 1*2 n

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PCR Refresher

Denature Anneal Extend

~95°C ~55-65°C ~72°C

Start # Cycle1 Cycle2 Cycle3 Cycle4 Cycle5 Cycle6 Cycle n

1 2 4 8 16 32 64 1*2 n

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PCR Refresher

Denature Anneal Extend

~95°C ~55-65°C ~72°C

Start # Cycle1 Cycle2 Cycle3 Cycle4 Cycle5 Cycle6 Cycle n

1 2 4 8 16 32 64 1*2 n

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PCR Refresher

Denature Anneal Extend

~95°C ~55-65°C ~72°C

Start # Cycle1 Cycle2 Cycle3 Cycle4 Cycle5 Cycle6 Cycle n

1 2 4 8 16 32 64 1*2 n

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Traditional PCR
Dynamics of amplification

Linear plot: Cycle v Copy #

7.0E+07
6.0E+07
5.0E+07
Copy #

4.0E+07
3.0E+07
2.0E+07
1.0E+07
0.0E+00
0 10 20 30 40
Cycle

Start # Cycle1 Cycle2 Cycle3 Cycle4 Cycle5 Cycle6 Cycle n

1 2 4 8 16 32 64 1*2 n

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Endpoint PCR
Analyze reactions at plateau

Plateau

Endpoint PCR
• Assayed at plateau
• Not quantitative
• Size & number
• Limited by gel sensitivity
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Semi-Quantitative PCR
Analyze reactions at an intermediate cycle

Semi-quantitative PCR
• Assayed before plateau
• Not linear
• Limited dynamic range

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Real-time PCR
Measure product at every cycle!

Taq
dNTPs
MgCl2
Buffer
Primers
Template

Fluorescent DNA
marker
Real-time PCR, quantitative PCR, qPCR
• Assayed at every cycle • Quantitative
• Requires specialized instrument • High-throughput capable
• No additional sample handling • Multiplex capable
• Broad dynamic range (106-108) • Typically used for <250bp
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Think About the Data

QUESTION 1: Which sample has a higher target concentration? Explain.

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Think About the Data

QUESTION 1: Which sample has a higher target concentration? Explain.

Fewer cycles
are required to
reach a given
copy #

Amplification is inversely proportional to starting concentration!

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Viewing the Data

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Viewing the Data

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Viewing the Data
Linear plot: Cycle v Fluorescence
7.0E+07
6.0E+07
5.0E+07

Fluorescence
4.0E+07
3.0E+07
Quantification is most 2.0E+07

robust in the exponential 1.0E+07 Exponential

phase of amplification 0.0E+00
0 10 20 30 40
Cycle

Semi-log plot: Cycle v Fluorescence

100,000,000
The exponential phase is 10,000,000
1,000,000
best viewed in a semi-log
Fluorescence

100,000
plot 10,000
1,000
100
10
1
0 10 20 30 40
Cycle

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From Raw Data to Quantification
Software processing

Step 0: Normalize to passive reference (Rn, normalized RFU)

• Only required on some platforms
• Use a second fluorescent dye, not influenced by DNA
• Divide reporter dye fluorescence by passive reference
Step 1: Subtract background fluorescence (RFU, ΔR, dR, dRn)
• Appropriate background cycles determined automatically
• Average background fluorescence for each reaction
• Subtract avg background fluorescence at each cycle
• De-trend data based on background fluorescence slope

Before After

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From Raw Data to Quantification
Software processing

Step 0: Normalize to passive reference (Rn, normalized RFU)

• Only required on some platforms
• Use a second fluorescent dye, not influenced by DNA
• Divide reporter dye fluorescence by passive reference
Step 1: Subtract background fluorescence (RFU, ΔR, dR, dRn)
• Average background fluorescence for each reaction
• Subtract avg background fluorescence at each cycle
• De-trend data based on background fluorescence slope
• Appropriate background cycles determined automatically
Step 2: Fit a curve to the data
Step 3: Determine a value (Cq) for each sample

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From Raw Data to Quantification
Quantification Cycle, Cq
Threshold method
• Cycle at which fluorescence crosses threshold
• Threshold set at ~10 StDev above background
• Threshold should be drawn in exponential phase
• Threshold variable between assays; must analyze separately!
RFU

Cq
amplification
threshold

Cycles
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From Raw Data to Quantification
Quantification Cycle, Cq
Threshold method
• Cycle at which fluorescence crosses threshold
• Threshold set at ~10 StDev above background
• Threshold should be drawn in exponential phase
• Threshold variable between assays; must analyze separately!

Cq amplification
threshold
RFU

Cycles
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From Raw Data to Quantification
Quantification Cycle, Cq
Regression method, or 2nd derivative maximum
• Point of maximum fluorescence increase
• Independent of other wells or assays on plate
• More consistent from plate to plate

Cq
RFU

Cycles
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 Real-Time PCR Chemistries &
Instrumentation

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Leading Real-Time PCR Chemistries

Dye-based Probe-based

Binds double stranded DNA Labelled probe

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 Dye-based qPCR

Uses a dsDNA-binding dye

• Low fluorescence of unbound dye
• Dye binds dsDNA stoichiometrically
• Measures all dsDNA in reaction
• Existing PCR assays can easily be
adapted*
• Less expensive
• Not multiplexed
• Allows melt analysis for QC or
genotyping

Examples:
SYBR Green, BRYT Green, LC Green, Eva Green
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Melt Analysis: An internal QC
Melt or Dissociation Curve
• After amplification, product is heated slowly, signal is continually measured
• As dsDNA amplicon denatures, dsDNA dye is displaced, signal decreases
• Melt peak ≈ Tm of product
• Impacted by # of amplicons, size & base composition

PCR Melt

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Melt Analysis: An internal QC
Melt or Dissociation Curve
• After amplification, product is heated slowly, signal is continually measured
• As dsDNA amplicon denatures, dsDNA dye is displaced, signal decreases
• Melt peak ≈ Tm of product
• Impacted by # of amplicons, size & base composition

RFU vs T(°C) dRFU/dT vs T(°C)

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 Melt Analysis: An Internal QC
Quality control

Single cDNA Primer dimer, low Tm

product gDNA
Multiple products

mut WT
mixture

High Resolution Melt (HRM)

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 Probe-based qPCR

Uses primers and 1-2 dye-labeled probes

• Increases specificity of target detection
• gDNA, psuedogenes still detected if amplified
• More expensive
• Allows for detection of multiple targets
• May require more optimization
• TaqMan® doesn’t allow melt analysis

Examples
• TaqMan® (hydrolysis probe)
• Molecular Beacons® (stem-loop FRET probe)
• Dual Hybridization Probes (donor-acceptor probe)
See Real-time PCR Detection Chemistries, Navarro et al. (2015)
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Probe-based qPCR:
TaqMan Fluorogenic 5’ Nuclease Assay

Quenched
hv
hydrolysis probe
• Probe contains reporter R Q
fluorophore and quencher primer

• Intact probe is quenched

• Taq degrades quenched probe
R
during extension Q
• Requires 5’ nuclease activity of
Taq
hv
• Irreversibly releases reporter Hydrolysis of probe
R results in fluorescence
dye from quenching
Q

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Multiplex Analysis
Analysis of multiple targets in the same well

Target 1
• Each target labeled with a different fluorophore Target 2
• Requires fewer wells for the same amount of data Single target

• Requires less sample

• Better normalization

• Same sample is assayed for all targets

• Requires more assay design & optimization Multiple targets

• Limited by instrumentation and dyes

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qPCR Chemistries
Dye-based vs. Probe-based

Dye-based qPCR Probe-based qPCR

Amplicon labeling dsDNA-binding dye Fluorescently labeled probes
Cost Lower cost Higher cost
Flexibility All optimized assays Single assay
Instrumentation All qPCR instruments Must match probes to filters
Specificity Measures all dsDNA Measures amplicon with probe
sequence
Multiplexing No Yes – different dyes/filters
Melt analysis Yes No (TaqMan)
QC and genotyping
Throughput High Highest (multiplexed)
Sample required Low Lowest (multiplexed)
Requires validation Yes Yes

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Reverse Transcription qPCR (RT-qPCR)
RT Taq
RNA

cDNA – no introns

Reverse Transcribe PCR

Applications Reverse Transcriptase

• Gene expression • RNA-directed DNA polymerase
• Biomarker discovery • Requires priming, Mg2+ or Mn2+ cofactor
• RNA Sequencing • RNase H activity in wildtype
• RNA viruses • Inhibits Taq polymerase
• cDNA cloning • AMV, MMLV
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Reverse Transcription
Two-step RT-qPCR—Making a pool of cDNA
Two Step RT-qPCR
Reverse Transcription
Heat denature RNA w/ primers
Add RT, Buffer, dNTPs & RNasin
Anneal & extend
Heat inactivate RT

Target mRNA
5’ AAAn
PCR or qPCR
Gene-specific primers Oligo dT Primed
cDNA
5’ AAAn

PCR Primers
Random Primed
cDNA
5’ AAAn

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Reverse Transcription
One-step RT-qPCR—Amplification of a single target
One Step RT-qPCR

RT & qPCR
Set up as for qPCR
Add RT and RNasin
Use gene-specific primers

Cycling considerations
Perform RT first
Inactivate RT/ Activate Taq
Standard qPCR cycling

Benefits
Uses less sample
Replicates over both steps
Quant & QC for FFPE RNA

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Real-Time Instruments

Examples:

Bio-Rad ABI Roche Stratagene

CFX96 Touch 7500 Fast LightCycler 480 Mx3005p

Hardware Differences Software Differences

• Excitation source (lamp, LED, LASER) • Analysis methods
• Detection method (CCD camera, PMT) • Analysis flexibility
• Filters (Filter wheel, # of filters) • Ease of use
• Specialized (Gradient block, # of wells, • Specialized (HRM, bar-coding, etc.)
interchangeable blocks, fast cycling) • Traceability, in vitro Diagnostic use

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Introduction to Quantification

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Comparing Samples
Assuming identical input amounts

Sample Copy# Cycle1 Cycle2 Cycle3 Cycle4 Cycle5 Cycle6 Cycle n

A 1 2 4 8 16 32 64 1*2 n
B 10 20 40 80 160 320 640 10*2 n

Quantity at a given cycle “n” is expressed as 𝒂 ∗ 𝟐𝒏

where 𝒂 is starting quantity

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 Comparing Samples
Assuming identical input amounts

ΔCq method
𝑏
= 2∆𝐶𝑞
𝑎
Not recommended!

Sample A will reach the threshold T = 𝑎 ∗ 2𝑥 at cycle 𝑥

Sample B will reach the threshold T = 𝑏 ∗ 2𝑦 at cycle 𝑦
Since the threshold T is the same for both,
𝑎 ∗ 2𝑥 = 𝑏 ∗ 2𝑦
If we rearrange to determine fold difference in starting quantities,
𝑏 2𝑥 2∗2∗2∗2∗⋯𝑥 𝑡𝑖𝑚𝑒𝑠
= = 2∗2∗2∗2∗⋯𝑦 𝑡𝑖𝑚𝑒𝑠 = 2𝑥−𝑦 = 2𝛥𝐶𝑞
𝑎 2𝑦

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Common Assumptions in Calculations

• ΔCq method
• ASSUMPTION 1: Amplification efficiency is perfect

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Common Assumptions in Calculations

ΔCq = 5

𝒃
QUESTION 2: What is the difference in target = 𝟐∆𝑪𝒒 = 𝟐𝟓
concentration between samples A & B if the 𝒂
assay has 100% amplification efficiency? 32-fold different
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Common Assumptions in Calculations

ΔCq = 5

𝒃
QUESTION 3: What is the difference in target = 𝟏. 𝟖∆𝑪𝒒 = 𝟏. 𝟖𝟓
concentration between samples A & B if the 𝒂
assay has 80% amplification efficiency? 18.9-fold different
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Common Assumptions in Calculations

• ΔCq method
• ASSUMPTION 1: Amplification efficiency is perfect
• Instead of 2 𝛥𝐶𝑞 , measure the assay efficiency experimentally and use it!
• Use your samples (or very similar ones) to determine efficiency
• ASSUMPTION 2: Identical inputs were used from each sample
• Quantification accuracy really matters!
• Normalize to a reference gene that is expressed at a constant concentration

• Δ Δ Cq method
𝑏
• Normalize between GOI and reference gene in the same sample
𝑎
• ASSUMPTION 3: Reference gene expression is constant
• Use multiple reference genes and verify expression is constant
• See Hellemans & Vandesompele (2014)!

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 The MIQE Guidelines—Read BEFORE You Plan!
The MIQE Guidelines
Minimum Information for Publication of Quantitative
Real-Time PCR Experiments
Bustin, et al., Clinical Chemistry 55:4, 611–622 (2009)

• Goal is to improve repeatability between labs

• Outlines common vocabulary to use
• Includes a checklist for reporting & planning
• Describes minimal reporting requirements
1. Description of sample manipulation
storage, extraction, quantification, integrity, etc.
2. Description of assay reagents, protocols, and controls
standard curves, ≥2 reference genes, -RT controls, NTCs
3. Assay validation—Required even for purchased assays!
sensitivity, specificity, and efficiency; reference gene stability
4. Description of calculations
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Real-Time PCR Resources On-line

General Real-Time PCR:

MIQE Guidelines: Bustin et al., Clin Chem (2009)
http://miqe-press.gene-quantification.info/
Gene Quantification: www.gene-quantification.info
Real-time chemistries: Navarro et al., Clinica Chimica Acta (2015)
Reference genes: Hellemans and Vandesompele, Methods Mol Biol (2014)
http://www.gene-quantification.com/Bio-Rad_2008_
Rethink_PCR_Conference_Hellemans_Vandesompele.pdf
geNorm and qBase+ software

Primer design software & Pre-designed assays:

Primer3 - http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi
Primer-BLAST - http://www.ncbi.nlm.nih.gov/tools/primer-blast/
IDT PrimerQuest - http://biotools.idtdna.com/Primerquest/
Primer Bank - http://pga.mgh.harvard.edu/primerbank/
RTPrimerDB - http://medgen.ugent.be/rtprimerdb
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Technical Services Scientists Ready to Help

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 Questions Welcome

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