Dr. Chaim WachtelApril 11, 2013

Introduction to Real-Time PCR

Real-Time PCR

• What is it?• How does it work• How do you properly perform an

experiment• Analysis

Michael Smith

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".

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 thatis proportional to the initial amount of DNA template.

• Comparison with standard curve gives quantification.

Real-Time and End PointEnd 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

RetractionWE WISH TO RETRACT OUR RESEARCH ARTICLE “THE MRNA OF THE ARABIDOPSIS GENE FT MOVESfrom 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 inthe 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

“Fifty Years of Molecular Diagnostics” Clin Chem. 2005 Mar;51(3):661-71 (C.Wittwer, ed.)

First real-time PCR, 1991

PCR tube in thermocycler

spectrofluorometerfiberoptic

First commercial real-time PCR instruments

ABI 7700 – laser/fiberoptic-based

ABI 5700 – CCD camera-based

Idaho Technology LightCycler – capillary tubes

7900HT Fast Real-Time PCR System(Sol Efroni’s lab)

RT-PCR machines at Bar Ilan

AB StepOnePlus Fast Real-Time PCR System

Qiagen’s Rotor-gene (Oren Levy’s lab) Bio-Rad CFX-96 Thermo PikoReal

(Bachelet Lab)

Rotor-gene

Probing alternativesNon-specific detection Dyes: SYBR Green I, BEBO,

BOXTO, EvaGreen...

Specific detection TaqMan probe Molecular Beacon Light-Up probe Hybridization probes

Primer based detection

Scorpion primers QZymeLux primers

SYBR Green binds to dsDNA

SYBR Green binds to DNA, particularly to double-stranded DNA, giving strongly enhanced fluorescence.

SYBR Green is sequence-dependent!

Low flourescence

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

• Detection of two (or more) different target sequences in the same reaction.

0 5 10 15 20 25 30 35 40

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Fluo

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ence

Cycle number

qPCR technical workflow

Sampling

DNA Extraction

RNA Extraction

DNase treatment

ReverseTranscription

qPCR DataAnalysis

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

Genomic DNA

Plasmid DNA

mRNA

Total RNA Fragment DNANuclear

RNA Phage DNAReverse

Transcription

Real-time PCR

DNARNA

Purification

Isolation

Disruption

AccessibilitySample 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)

• Con:– Higher DNA

contamination (for RNA isolation)

– Separate DNase I digestion with additional purification

Spin columns• Pro:

– Less contaminating DNA (for RNA isolation)

– On column DNase digestion Less loss of RNA

– Higher quality– Easy to use

• 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/RNA-

binding 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 Bad RNA Quality10 RNA Quality Indicator 1

Publications on RNA integrity

DNase I treatment of RNA samples

No RT, DNase

RT, DNase

RT, No DNase No RT, No DNase

qPCR technical workflow

Sampling

DNA Extraction

RNA Extraction

DNase treatment

ReverseTranscription

qPCR DataAnalysis

Reverse transcription RT

Outline

• Priming efficiency

• Reproducibility

• Properties of Reverse transcriptase

• RNA concentrations

General description of RT reaction

Reverse Transcriptases are RNA-dependent* 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

Hexa

me

r

Olig

o(d

T)

Mix

B-tubulin

Ca

V1D

Gap

dh

Insu

lin2

Glu

t2

No p

rimer

RNA pool

RT priming

RT replicate (D = 5, n =5)RT RT

QPCR replicate (D =2, n =10)QPCR QPCR

Figure 1

Real-time PCR with different RT primers

0 10 20 30 40 500

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50

specific primeroligo(dT)

random hexamer

non-priming

Flu

ores

cenc

e

Cycle number

Dependence on priming strategy

Btubulin CaV1D Gapdh Ins II Glut20

2

4

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14

16

RT

effi

cie

ncy

Rand hex Oligo(dT) Gene specific Mixture gene specific

Dependance of priming method

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

max DCt 1,4 2,3 0,8 1,5 4,4

Gene

RT p

rim

ing

meth

od

Specificity of specific priming

b-tubulin

CaVID

GAPDH

Insulin II

Glut 2

b-tubulin 18,8 28,7 19 18,8 30,6

CaVID 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 32,6NTC ~ 35

PCR primers used

RT p

rim

ers

use

d

37ºC

GAPDH 3’

60ºC

18 unpaired bases

24 unpaired bases

Algorithm: mfold

Comparison of reverse transcriptases

Temp

Ref: Ståhlberg et al. Comparison of reverse transcriptases in gene expression analysis.

Clin.Chem. 50(9); 1678-1680 (2004)

MMLV RNase H- Minus (Promega, Germany)37

M-MLV (Promega)45

Avian Myeloblastosis Virus (AMV) (Promega) 37

Improm-II (Promega)45

Omniscript (Qiagen, Germany)37

cloned AMV (cAMV) (Invitrogen, Germany)45

ThermoScript RNase H- (Invitrogen) 50

SuperScript III RNase H- (Invitrogen) 50

100 – fold variation in RT yield

MMLV MMLVH AMV Improm Omni cAMV Thermo Super

35

40

*

*

*

*

*

HTR2a

Ct

8 transcriptases tested on 6 genes

MMLV MMLVH AMV Improm Omni cAMV Thermo Super

20

25

*

* -actin

Ct

MMLV MMLVH AMV Improm Omni cAMV Thermo Super20

25

30

GAPDH

Ct

MMLV MMLVH AMV Improm Omni cAMV Thermo Super

35

40

*

*

*

*

*

HTR2a

Ct

MMLV MMLVH AMV Improm Omni cAMV Thermo Super20

25

30

HTR1a

Ct

MMLV MMLVH AMV Improm Omni cAMV Thermo Super20

25

30

HTR1b

Ct

MMLV MMLVH AMV Improm Omni cAMV Thermo Super20

25

30

*

HTR2b

Ct

Experimental design to study linearity

RNA pool Yeast tRNA or water

1024

ng

256

ng

64 n

g

16 n

g

4 ng

RT replicate (D = 2, n =2)RT RT

QPCR replicate(D = 2, n =4)QPCR QPCR

Effect of carrier

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1024 ng256 ng64 ng16 ng

A

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nce

Cycle number

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B

1024 ng256 ng64 ng16 ng4 ng

Flu

ores

cenc

eCycle number

+ tRNA- tRNA

Effect of carrier

10 100 100014

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34A

Glut2 CaV1D -tubulin InsulinII Gapdh

Ct

Total RNA (ng)

2x107

2x106

2x105

2x104

2x103

2x102

cD

NA

mol

ecul

es

10 100 1000

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Glut2 CaV1D -tubulin InsulinII Gapdh

Ct

Total RNA (ng)

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102C

cD

NA

mol

ecul

es

+ tRNA- tRNA

RNA dilutions

10 100 100014

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34A

Glut2CaV1D-tubulinInsulinIIGapdh

Ct

Total RNA (ng)

2x10

2x10

2x10

2x10

2x10

2x10

cDNA

molec

ules

10 100 1000

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Glut2CaV1D-tubulinInsulinIIGapdh

Ct

Total RNA (ng)

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B

cDNA

molec

ules

10 100 1000

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Glut2CaV1D-tubulinInsulinI IGapdh

Ct

Total RNA (ng)

1 0

1 0

1 0

1 0

1 0

1 0C

cDNA

molec

ules

10 100 1000

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Glut2CaV1D-tubulinInsulinIIGapdh

Ct

TotalRNA (ng)

1 0

1 0

1 0

1 0

1 0

1 0D

cDNA

mol

ecules

Figure 2

Water

Yeast tRNA

Oligo(dT) Random Hexamers

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