BCH5000 Real-time PCR Workshop 2006

7/28/2019 BCH5000 Real-time PCR Workshop 2006

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Real-Time PCR

Workshop

1. Why Real-time PCR? Advantages and Disadvantages

2. Theory of Real-time PCR

3. Types of Real-time PCR Quantification

4. Choosing Housekeeping Gene for Normalization

All information are adapted from relevant websites

as qPCR using real-time detections are developed

by different companies. Please read carefully and

follow protocols from the kits you use.


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Disadvantage of traditional PCR* Poor precision (Northern could even be better)

* Low sensitivity

* Short dynamic range < 2 logs* Low resolution

* Non-automated

* Size-based discrimination only

* Results are not expressed as numbers* Ethidium bromide staining is not very quantitative

* Competitive (mimic) PCR is laborious and tedious(very labor intensive)

ABI: Real-Time PCR vs Traditional PCR (www)

1. Why Real-time PCR ?
http://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdfhttp://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf


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wider dynamic range



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1. Requires expensive equipments and reagents

2. Intra- and inter-assay variations

3. Due to its extremely high sensitivity, you may get

high deviations of the same treatment, optimal

benefit from the above advantages requires a

clear understanding of the many options available

for running real-time PCR experiment:

theory of real-time PCR

Disadvantages of real-time PCR



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2. Theory of real-time PCR


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Linear ground phase:

PCR is just began

Fluorescence emission at each cycle has not yet risen above background

Baseline fluorescence is calculated at this time

CT - threshold cycle:

the first significant increase in

the amount of PCR product

correlates to the initial amount of

target template

CTrepresents the starting copy

no. in the original template

Early exponential phase:

PCR is just began

The amount of fluorescence has reached a threshold where it is

significantly higher than background (usually 10 times the standarddeviation of the baseline)

PCR can be broken into 4 major phases



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Log-linear phase:

PCR reaches its optimal amplification period with the

PCR doubling after each cycle in ideal reaction conditions

Plateau phase:

The plateau stage is reached when reaction components become limited

and the fluorescence intensity is no longer useful for data calculation



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The five-fold dilution series seems to plateau at the same place even though the exponentialphase clearly shows a difference between the points along the dilution series. This reinforcesthe fact that if measurements were taken at the plateau phase, the data would not trulyrepresent the initial amounts of starting target material.



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The Amplification Plot contains valuable information for the quantitative measurement of DNA orRNA. The Threshold line is the level of detection or the point at which a reaction reaches a

fluorescent intensity above background. The threshold line is set in the exponential phase of theamplification for the most accurate reading. The cycle at which the sample reaches this level is

called the Cycle Threshold, CT. These two values are very important for data analysis using the 5nuclease assay.

What is CT (threshold cycle)?2. Theory of Real-time PCR


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0 10 20 30 40cycle number

Rn

CT

Threshold Rn

Sample

No Template

Control

What is Rn?

-Rn

+Rn

1. Rn+ is the Rn value of a reaction containing all components (the

sample of interest);

2. Rn- is the Rn value detected in NTC (baseline value)

3. DRn is the difference between Rn+ and Rn-. It is an indicator of themagnitude of the signal generated by the PCR

4. DRn is plotted against cycle numbers to produce the amplification

curves and gives the CT value



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3.Types of real-time PCRquantificationa: Detection

b: Calculation

c: Normalization (in part4 )


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Detection chemistries

Four common ways:1.DNA binding dyes

E.g. SYBR Green

2.Hydrolysis probes

TaqMan3.Hybridisation probes

E.g. Light cycler

4.Hairpin probes

Molecular beacons

3. PCR Quantification


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SYBR Green (double-stranded DNA binding dye)At the beginning of amplification, the reaction mixture contains the denatured

DNA, the primers, and the dye. The unbound dye molecules weakly fluoresce,

producing a minimal background fluorescence signal which is subtracted

during computer analysis.(1) After annealing of the primers, a few dye molecules can bind to the double

strand. DNA binding results in a dramatic increase of the SYBR Green I

molecules to emit light upon excitation.

(2) During elongation, more and more dye molecules bind to the newly

synthesized DNA. If the reaction is monitored continuously, an increase in

fluorescence is viewed in real-time. Upon denaturation of the DNA for the nextheating cycle, the dye molecules are released and the fluorescence signal falls.

Mapping Protein/DNA Interactions by Cross-Linking (NCBI Books) (www)


C Q f
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=inserm.section.325http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=inserm.section.325


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SYBR Green* emits a strong fluorescent signal upon binding

to double-stranded DNA

* nonspecific binding (primer dimer) is a

disadvantage; check your reactions on gel first!

* requires extensive optimisation

* requires melt ing point cu rve determ inat ion

* longer ampl icons c reate a stronger signal,

ampl icons shou ld be 100 to 200 bp in size


3 PCR Q tifi ti


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* Assays that do not require specificity of probebased assays. Detection of 1000s of molecules

* When the PCR system is fully optimized -no primer

dimers or non-specific amplicons, e.g. from

genomic DNA

When to choose SYBR Green

When NOT to choose SYBR Green

* Allelic discrimination assays

* Multiplex reactions

* Amplification of rare transcripts* Low level pathogen detection from

environmental samples or limited samples



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Mocellin et al. Trends Mol Med 2003(www)

The TaqMan 5 exonuclease assay

FRET =

Frster/fluorescence

resonance energy

transfer

&

DNA Polymerase 5'

exonuclease activity

http://dx.doi.org/doi:10.1016/S1471-4914(03)00047-9http://dx.doi.org/doi:10.1016/S1471-4914(03)00047-9


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FRET

ABI: Real-Time PCR vs Traditional PCR(www)


Th T M 5 l
http://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdfhttp://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf


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The TaqMan 5 exonuclease assay

In addition to two conventional PCR

primers, P1 and P2, which are specific for the

target sequence, a third primer, P3, is

designed to bind specifically to a site on

the target sequence downstream of the P1

binding site. P3 is labelled with two

fluorophores, a reporter dye (R) is

attached at the 5 end, and a quencher

dye (D), which has a different emissionwavelength to the reporter dye, is attached at its 3

end. Because its 3 end is blocked, primer P3

cannot by itself prime any new DNA synthesis.

During the PCR reaction, Taq DNA polymerase

synthesizes a new DNA strand primed by P1 and

as the enzyme approaches P3, its 5 3

exonuclease activity processively degrades the P3

primer from its 5 end. The end result is that thenascent DNA strand extends beyond the

P3 binding site and the reporter and

quencher dyes are no longer bound to the

same molecule. As the reporter dye is no longerin close proximity to the quencher, the resulting

increase in reporter emission intensity is easilydetected.

Human Molecular Genetics 2. NCBI Books (www)


Probe sequences are not altered by PCR so they can still be used in a
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=hmg.chapter.551http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=hmg.chapter.551


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A The donor-dye probe is labeled with fluorescein at the

3 end and the acceptor-dye probe is labeled with

LightCycler Red at the 5 end. Hybridization does not

take place during the denaturation phase of PCR and,thus, the distance between the dyes is too large to allow

energy transfer to occur.

B During the annealing phase, the probes hybridize to

the amplified DNA fragment in a close head-to-tail

arrangement. When fluorescein is excited by the lightfrom the LED, it emits green

fluorescent light, transferring the energy to LightCycler

Red, which then emits red fluorescent light. This red

fluorescence is measured at the end of each annealing

step, when the fluorescence intensity is highest.

C After annealing, the temperature is raised and the

HybProbe probe is displaced during elongation. At the

end of this step, the PCR product is double-stranded

and the displaced HybProbe probes are again too far

apart to allow FRET to occur.


Probe sequences are not altered by PCR, so they can still be used in a

subsequent assay, e.g., for mutation detection or SNP analysis

https://www.roche-applied-science.com/sis/rtpcr/htc/htc_fst/010500.jsp

Light cycler


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Wittwer, 1997 (www)

Comparing three different fluorescence-monitoring systemfor DNA amplification.

Depends on theaccumulated

amplification product

Depends on the 5-

exonuclease activity of

the polymerase

Depends on the

independent

hybridization of

adjacent donar and

acceptor probes

http://www.idahotech.com/product_sup/articles/continuious/fig1.htmhttp://www.idahotech.com/product_sup/articles/continuious/fig1.htm


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Mocellin et al. Trends Mol Med 2003(www)

Molecular Beacons

1. Molecular beacons are thesimplest hairpin probeflanked by 2 invertedrepeats.

2. Reporter and quencherdyes are attached to each

end of the molecule,causing a reduction influorescence emission inhairpin formation

3. When bound to the target,the quencher and reporter

are separated, allowingreporter emission.

Thermodynamic stability:

Probe-target helix > hairpin structure > mis-matched probe target helix

http://dx.doi.org/doi:10.1016/S1471-4914(03)00047-9http://dx.doi.org/doi:10.1016/S1471-4914(03)00047-9


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* Absolute quantification* Relative quantification (relative fold change)

i. Relative standard methodii. Comparative CT (2

-CT) method

3. Types of real-time PCR

quantification



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*Absolute quantification

* Relative quantification (relative fold change)Relative quantification determines the changes in

steady-state mRNA levels of a gene across multiple

samples and expresses it relative to the levels o f an

internal con tro l RNA.

relative quantification does not require standards with

known concentrationsi. Relative standard method

ii. Comparative CT (2-CT) method

3. Types of real-time PCR

quantification



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Relative standard curve method

1. Construct a relativestandard curve

2. Calculate the input amount by entering

the following formula in an adjacent cell:

= 10^ [cell containing log input amount]

3. Divide the amount of c-myc bythe amount of GAPDH to

determine the normalized amount

of c-myc (c-mycN).



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* Absolute quantification

* Relative quantification

(relative fold change vscalibrator)i. Relative standard curve method

ii. Comparative CT (2-CT) method

Types of real-time PCR

quantification


Validation experiment for comparative C method


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ABI-7700 User Bulletin #2

Validation experiment for comparative CT method

3. PCR uantification

Comparative C method I


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Comparative CT method-I




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Comparative CT method - II



3 PCR Quantification


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targettreatedref treated

targetcontrol

ref control

av =19.80

av =19.93

av =18.03

av =29.63

Ct = 9.70

Ct = -1.7

Ct = target - ref

Ct = target - ref

Difference = Ct-Ct= Ct= (-1.7) -9.70= -11.40

control

experiment

Exercise: By 2 CT, fold change=??? 2702



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4. Housekeeping Gene for Normalization

Housekeeping Gene for Normalization

1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

2. b -actin

3. Ribosomal RNA (rRNA): 28S, 18S

None of the identified reference for data normalization are ideal.

GAPDH


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GAPDH

(Bustin, 2000)4. Housekeeping Gene for Normalization



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GAPDH concentrations vary between different individuals (Bustin et al. 1999),

during pregnancy (Cale et al. 1997),

with developmental stage (Puissant et al. 1994, Calvo et al. 1997), during the cell cycle (Mansuret al. 1993),

apoptosis (Ishitani et al. 1997)

food deprivation (Yamada et al. 1997)

*** Inducer

after the addition of the tumour promoter 12-Otetradecanoyl-phorbol-13-acetate (Spanakis1993),dexamethasone (Oikarinen et al. 1991) and carbon tetrachloride (Goldsworthy et al.

1993). Insulin stimulates GAPDH transcription (Rolland et al1995, Barroso et al. 1999)

calcium ionophore A23187 induces GAPDH transcription

Growth hormone (Freyschuss et al. 1994), vitamin D (Desprez et al. 1992), oxidativestress (Ito et al. 1996), hypoxia (Graven et al. 1994, Zhong & Simons 1999), manganese(Hazell et al. 1999) and the tumour suppressorTP53 (Chen et al. 1999), have all beenshown to activate its transcription

retinoic acid (Barroso et al. 1999) downregulate GAPDH transcription in the gut and inadipocytes, respectively.

*** unregulated in cancer

in rat hepatomas (Chang et al. 1998),

Malignant murine cell lines (Bhatia et al. 1994) and

Human prostate carcinoma (Ripple & Wilding 1995)

Its use as an internal standard is inappropriate

It is a mystery why GAPDH continues to find favor as an internal standard.

p g


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b-actin concentrations vary widely in

response to

experimental manipulation in human breast epithelialcells (Spanakis 1993)

in various porcine tissues (Foss et al. 1998) caninemyocardium (Carlyle et al. 1996)

the presence ofpseudogenes interferes with theinterpretation of results (Dirnhoferet al. 1995, Raffet al.1997, Mutimeret al. 1998)

primers commonly used for detecting -actin mRNAamplify DNA (Dakhama et al. 1996).



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Common normalizing

housekeeping gene

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

b -actin

Ribosomal RNA (rRNA)

28S, 18SVarying ratios of rRNA to mRNA have been

reported (Solanas et al.,2001)

Use random hexamer instead of oligo dT inthe RT step



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Optimization of Primers

* equal Tm (58-600 C)

* 15-30 bases in length

* G+C content 30-80%

* no runs of four or more Gs (any nucleotide)

* no more than two G+C at the 3 end

* no G at the 5' end

* amplicon size 50-150 bp (max 400)

* span exon-exon junc t ions in cDNA to avoid

genomic DNA being amplified

ABI Primer Express Software Tutorial (www)

http://www.appliedbiosystems.com/support/tutorials/pdf/taqman_mgb_primersprobes_for_gene_expression.pdfhttp://www.appliedbiosystems.com/support/tutorials/pdf/taqman_mgb_primersprobes_for_gene_expression.pdf


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References

1. Wong, M.L. and Medrano J.F. 2005 Real-time PCRfor mRNA quantitation. Biotechniques 39(1): 75-85.

2. Bustin, S.A. 2002 Quantification of mRNA using real-

time reverse transcription PCR (RT-PCR): trends and

problems. J Mol Endocrinol29:23-39.3. Bustin, S.A. 2000 Absolute quantification of mRNA

using real-time reverse transcription polymerase chain

reaction assays. J Mol Endocrinol25:169-193.

4. Websites from ABI, MJ companies, etc.

Documents

BCH5000 Real-time PCR Workshop 2006