Introduction to Real-Time
PCR
Dr. Chaim Wachtel
April 11, 2013
Real-Time PCR
• What is it?
• How does it work
• How do you properly perform an
experiment
• Analysis
The Nobel Prize in Chemistry 1993 was awarded "for contributions to the
developments of methods within DNA-based chemistry" jointly with one half
to Kary B. Mullis "for his invention of the polymerase chain reaction (PCR)
method"and with one half to Michael Smith "for his fundamental
contributions to the establishment of oligonucleotide-based, site-directed
mutagenesis and its development for protein studies".
Michael Smith
PCR – A simple idea
• Polymerase Chain Reaction: Kary Mullis (1983)
• In vitro method for enzymatically synthesizing
DNA
• The reaction uses two oligonucleotide primers that
hybridize to opposite strands and flank the target
DNA sequence that is to be amplified
• A repetitive series of cycles gives exponential
accumulation of a specific DNA fragment
– Template denaturation
– Primer annealing
– Extension of annealed primers by the polymerase
• The number of target DNA copies doubles every
PCR cycle (20 cycles  220≈106 copies of target)
Principle of PCR
Difference PCR vs real-time PCR?
• Fluorescence is
measured every cycle
(signal  amount of PCR
product).
• Curves rise after a
number of cycles that
is proportional to the
initial amount of DNA
template.
• Comparison with
standard curve gives
quantification.
Real-Time and End Point
End point
Real time
MIQE: the minimum
information
for the publication of qPCR
experiments.
http://www.rdml.org/miqe.php
The mRNA of the Arabidopsis Gene FT Moves from
Leaf to Shoot Apex and Induces Flowering
Tao Huang, Henrik Böhlenius, Sven Eriksson,
François Parcy, and Ove Nilsson
Science 9 September 2005: 1694-1696.
2005: Signaling Breakthroughs of the Year
Retraction
WE WISH TO RETRACT OUR RESEARCH ARTICLE “THE MRNA OF THE
ARABIDOPSIS GENE FT MOVES
from leaf to shoot apex and induces flowering” (1). After the first author
(T.H.) left the Umeå Plant Science Centre for another position, analysis of
his original data revealed several anomalies.
It is apparent from these files that data from the real-time RT-PCR were
analyzed incorrectly.
Certain data points were removed, while other data points were given
increased weight in
the statistical analysis.
When all the primary real-time RT-PCR data are subjected to correct
statistical analysis, most of the reported significant differences between
time points disappear.
Because of this, we are retracting the paper in its entirety.
Real-Time Machines
• How do they work
• What can you do with one
– Gene expression
– SNP detection
– DNA detection (quantify)
• How do you use them
– Experiment design
• Everything you need to know and more about
RNA and RT-PCR
First real-time PCR, 1991
spectrofluorometer
fiberoptic
PCR tube in
thermocycler
“Fifty Years of Molecular Diagnostics”
Clin Chem. 2005 Mar;51(3):661-71
(C.Wittwer, ed.)
First commercial real-time PCR
instruments
ABI 7700 – laser/fiberoptic-based
ABI 5700 – CCD camera-based
Idaho Technology LightCycler –
capillary tubes
RT-PCR machines at Bar Ilan
AB StepOnePlus
Fast Real-Time PCR System
7900HT Fast Real-Time PCR System
(Sol Efroni’s lab)
Qiagen’s Rotor-gene
(Oren Levy’s lab)
Bio-Rad CFX-96
Thermo PikoReal
(Bachelet Lab)
Rotor-gene
Probing alternatives
Non-specific detection
Dyes: SYBR Green I, BEBO,
BOXTO, EvaGreen...
Primer based detection
Scorpion primers
QZyme
Lux primers
Specific detection
TaqMan probe
Molecular Beacon
Light-Up probe
Hybridization probes
SYBR Green binds to dsDNA
SYBR Green binds to
DNA, particularly
to double-stranded
DNA, giving
strongly enhanced
fluorescence.
SYBR Green is
sequencedependent!
Low flouresc enc e
The TaqMan Probe
• The TaqMan probe
binds to ssDNA at a
combined annealing
and elongation step.
• It is degraded by the
polymerase, which
releases the dye from
the quencher.
Multiplex Q-PCR
40
Fluorescence
30
20
10
0
0
5
10
15
20
25
30
35
Cycle number
• Detection of two (or
more) different
target sequences in
the same reaction.
40
qPCR technical workflow
DNA
Extraction
Sampling
qPCR
RNA
Extraction
DNase
treatment
Reverse
Transcription
Data
Analysis
Nucleic acid isolation and
purification
Overview
• Sampling
• Accessibility and lysis
• Commonly used techniques
• RNA considerations
• Quality control
Why sample preparation?
• Make target available
• Remove inhibitors
• Remove fluorescent contaminants
• Preserve target integrity
• Concentrate target
Path
Disruption
Isolation
mRNA
Total
RNA
Nuclear
RNA
Purification
RNA
DNA
Reverse
Transcription
Real-time PCR
Genomic DNA
Plasmid DNA
Fragment DNA
Phage DNA
Accessibility
Sample disruption and homogenization
– Mechanical
• Grinding, Sonication, Vortexing, Polytron
– Physical
• Freezing
– Enzymatic
• Proteinase K, Lysozyme, Collagenase
– Chemical
• Guanidinium isothiocyanate (GITC), Alkali
treatment, CTAB
Lysis
– Complete or partial lysis?
– Chaotropic lysis buffers:
• SDS, GITC, LiCl, phenol, sarcosyl
– Gentle lysis buffers:
• NP-40, Triton X-100, Tween, DTT
Purification principles
• Characteristics of nucleic acids
– Long, unbranched, negatively charged polymers
• Examples:
– Differential solubility
– Precipitation
– Strong affinity to surface
• Factors:
– pH, [salt], hydrophobicity
Purification techniques
• Solution based- eg Tri reagent, CsCl gradient
• Precipitation- ethanol, needs salt, multiple factors
can influence precipitation
• Membrane based- spin columns (Qiagen and the
like)
• Magnetic bead based
Solution based isolation
• Most methods use hazardous reagents
• Phenol/Chloroform extraction
– Proteins, lipids, polysaccharides go into the
organic phase or in the interphase.
– DNA/RNA remains in aqueous phase
• Caesium chloride density gradient
ultracentrifugation
– Time consuming
• Acid guanidine phenol chloroform extraction
– Commonly called TRIzol
Precipitation purification
• Nucleic acids precipitate in alcohols
• Salt (NaCl, NaAc) facilitates the process
• Important factors: Temperature, time, pH, and
amount
Membrane based isolation
• Anion exchange technology
• Spin column / silica gel membrane
– Chaotropic salts (e.g. NaI or guanidine
hydrochloride) bind H2O molecules
– Loss of water from DNA changes shape and
charge
– DNA binds reversibly to silica membrane
Purification – GITC vs. column
Organic liquids
• Pro:
– Higher yield
– Can handle larger amounts
of cells
– Better for troublesome
tissues (fatty tissue, bone
etc)
Spin columns
• Pro:
– Less contaminating DNA
(for RNA isolation)
– On column DNase digestion
Less loss of RNA
– Higher quality
– Easy to use
• Con:
– Higher DNA contamination
(for RNA isolation)
– Separate DNase I
digestion with additional
purification
• Con:
– Limited loading capacity
– More expensive (?)
RNA Considerations
• RNA is chemically and biologically
less stable than DNA
• Extrinsic and intrinsic
ribonucleases (RNases)
– Specific and Nonspecific inhibitors
Stabilizing conditions
• Work on ice
• Process immediately
• Flash freeze sample in liquid nitrogen and store at
-70°C until later use
• Store samples in stabilization buffer
Storage of nucleic acids
• Nuclease-free plasticware
• Eluted in nuclease-free water, TE or sodium citrate
solution
• RNA:
• Neutral pH to avoid degradation
• Aliquot sample to avoid multiple freeze-thaw cycles
• Isolated RNA should be stored at -20 deg C or -70
deg C for even better protection in ethanol and not
water.
Quality Control
• Spectroscopic methods
– Concentration, [NA] = A260 x e mg/ml
– Purity: A260 / A280 (≈1.8 for DNA, 2.0 for RNA)
• Dyes
– Quantification by fluorescence of DNA/RNAbinding dyes (Qubit)
• Electrophoresis (28S and 18S bands)
What is the BioAnalizer?
• Microfluidic separations
technology
• RNA - DNA - Protein
• 1µl of RNA sample (100 pg to 500
ng)
• 12 samples analyzed in 30 min
• Integrated analysis software:
– Quantitation
– Integrity of RNA
Bioanalyzer
RNA Integrity: RQI
Good RNA Quality
10
RNA Quality Indicator
Bad RNA Quality
1
Publications on RNA integrity
DNase I treatment of RNA
samples
RT, No DNase
No RT, No DNase
RT, DNase
No RT, DNase
qPCR technical workflow
DNA
Extraction
Sampling
qPCR
RNA
Extraction
DNase
treatment
Reverse
Transcription
Data
Analysis
Reverse transcription RT
Outline
• Priming efficiency
• Reproducibility
• Properties of Reverse
transcriptase
• RNA concentrations
General description of RT
reaction
Reverse Transcriptases are RNAdependent* DNA polymerases that
catalyze first strand DNA synthesis in
presence of a suitable primer+ as long
as it has a free 3’ OH end.
*Can use also single strand DNA as template.
+ Can
be either RNA or DNA.
RT priming
RT with Gene-Specific Priming
RT with Oligo(dT) Priming
RT with Random Hexamer Priming
Real-time PCR using different RT
primers
No p rim er
Glut2
Insulin2
Gap dh
Ca V1D
B-tubulin
Mix
Olig o(d T)
Hexame r
RNA pool
RT priming
RTreplic ate
(DRT= 5, nRT= 5)
QPCR replicate
(DQPCR= 2, n QPCR= 10)
Real-time PCR with different RT
primers
50
45
40
Fluorescence
35
30
random hexamer
25
20
15
10
non-priming
oligo(dT)
specific primer
5
0
0
10
20
30
Cycle number
40
50
Dependence on priming strategy
16
14
Rand hex
Oligo(dT)
Gene specific
Mixture gene specific
RT efficiency
12
10
8
6
4
2
0
Btubulin
CaV1D
Gapdh
Ins II
Glut2
Dependance of priming method
RT priming method
Gene
b-tubulin
CaVID GAPDH
Insulin
II
Glut 2
hexamers
19,5
26,5
15,8
16,9
27,5
oligo dT
18,1
28,8
16,6
15,9
28,4
GSP
18,8
28,7
16,4
17,4
31,8
mix
19,1
27,9
16,3
16,6
29,3
1,4
2,3
0,8
1,5
4,4
max DCt
Specificity of specific priming
RT primers used
PCR primers used
Insulin
II
b-tubulin CaVID GAPDH
b-tubulin
Glut 2
18,8
28,7
19
27
18,7
19,9
22,8 -
GAPDH
23,4
30,1
16,4
20,1
29,7
Insulin II
23,5
31,6
20
17,4
31
Glut 2
25,8
31,9
22,7
22,7
31,8
no RT
primer
27,6
33,7
23,6
23,1
32,6
CaVID
NTC ~ 35
18,8
30,6
GAPDH 3’
60ºC
37ºC
24 unpaired bases
18 unpaired bases
Algorithm: mfold
Comparison of reverse transcriptases
Temp
MMLV RNase H- Minus (Promega, Germany)
M-MLV (Promega)
Avian Myeloblastosis Virus (AMV) (Promega)
Improm-II (Promega)
Omniscript (Qiagen, Germany)
cloned AMV (cAMV) (Invitrogen, Germany)
ThermoScript RNase H- (Invitrogen)
SuperScript III RNase H- (Invitrogen)
Ref: Ståhlberg et al. Comparison of reverse
transcriptases in gene expression analysis.
Clin.Chem. 50(9); 1678-1680 (2004)
37
45
37
45
37
45
50
50
100 – fold variation in RT yield
*
HTR2a
40
Ct
*
*
*
35
*
MMLV MMLVH
AMV
Improm
Omni
cAMV Thermo Super
8 transcriptases tested on 6 genes
30
*
GAPDH
-actin
*
40
*
*
Ct
Ct
Ct
25
25
HTR2a
*
*
35
*
20
20
MMLV MMLVH AMV Improm Omni
MMLV MMLVH AMV Improm Omni
cAMV Thermo Super
30
30
HTR1b
cAMV Thermo Super
MMLV MMLVH AMV Improm Omni
HTR1a
cAMV Thermo Super
30
20
20
MMLV MMLVH AMV Improm Omni
cAMV Thermo Super
*
25
Ct
25
Ct
Ct
HTR2b
MMLV MMLVH AMV Improm Omni
cAMV Thermo Super
25
20
MMLV MMLVH AMV Improm Omni
cAMV Thermo Super
Experimental design to study linearity
4 ng
16 ng
64 ng
256 ng
1024 ng
RNA pool
Yeast tRNA
or
water
RTreplic ate
(DRT = 2, nRT= 2)
QPCR replicate
(DQPCR = 2, nQPCR= 4)
Effect of carrier
B
A
20
30
18
25
16
Fluorescence
Fluorescence
14
12
10
8
1024 ng
256 ng
64 ng
16 ng
6
4
2
20
15
1024 ng
256 ng
64 ng
16 ng
4 ng
10
5
0
0
-2
0
10
20
30
Cycle number
- tRNA
40
50
60
0
10
20
30
Cycle number
+ tRNA
40
50
60
Effect of carrier
A
34
2
10
32
32
C
30
2
2x10
3
30
10
28
28
22
20
18
16
Glut2
CaV1D
-tubulin
InsulinII
Gapdh
5
2x10
6
2x10
4
10
24
22
20
18
5
10
Glut2
CaV1D
-tubulin
InsulinII
Gapdh
6
10
16
7
14
7
2x10
10
100
Total RNA (ng)
- tRNA
1000
10
10
100
Total RNA (ng)
+ tRNA
1000
cDNA molecules
4
2x10
24
26
Ct
Ct
26
cDNA molecules
3
2x10
RNA dilutions
Oligo(dT)
Random Hexamers
B
A
34
32
32
Water
10
2x10
30
30
10
2x10
Ct
24
20
18
16
Glut2
CaV1D
-tubuli n
InsulinII
Gapdh
2x10
22
20
2x10
14
10
100
1000
10
24
18
Glut2
CaV1D
-tubulin
InsulinII
Gapdh
10
10
10
32
10
32
D
30
10
Ct
Glut2
CaV1D
-tubulin
Insuli nII
Gapdh
10
10
16
26
10
24
Ct
10
24
cDNA molecules
28
26
18
1000
10
28
20
100
Total RNA (ng)
C
30
22
10
16
2x10
Total RNA (ng)
Yeast tRNA
10
22
20
18
10
Glut2
CaV1D
-tubulin
InsulinII
Gapdh
10
16
10
10
100
Total RNA (ng)
1000
10
10
100
Total RNA (ng)
1000
cDNA mol ecules
22
26
cDNA molecu les
26
28
Ct
2x10
cDNA molecules
28
Conclusions
•
•
•
•
•
•
•
•
The RT reaction shows higher technical variability than QPCR
There is no optimum priming strategy
Gene specific primers must target accessible regions
The RT yield changes over 100-fold with the choice of reverse
transcriptase
The yield variation is gene specific
RT yield is proportional to the amount of template in presence
of proper carrier
Typical RT yield is 10-50 %
RT-QPCR is highly reproducible as long as the same protocol and
reaction conditions are used
The efficiency of the RT reaction varies from gene to gene
and depends on the conditions – run the RT of all samples
using exactly the same protocol and reagents under the same
conditions