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.

1. Why Real-time PCR ?

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

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)

1. Why Real-time PCR ?

Advantages of real-time PCR

amplification can be monitored real-time wider dynamic range of up to 1010-fold 10,000 to 100,000-fold more sensitive than RNase protection assay, 1000-fold more sensitive than dot blot hybridization (i.e. requirement of 1000-fold less RNA than conventional assays) no post-PCR processing of products (No gel-based analysis at the end of the PCR reaction high throughput) ultra-rapid cycling (30 minutes to 2 hours) highly sequence-specific

1. Why Real-time PCR ?

wider dynamic range

1. Why Real-time PCR ?

Advantages of real-time PCR

amplification can be monitored real-time wider dynamic range of up to 1010-fold 10,000 to 100,000-fold more sensitive than RNase protection assay, 1000-fold more sensitive than dot blot hybridization (i.e. requirement of 1000-fold less RNA than conventional assays) no post-PCR processing of products (No gel-based analysis at the end of the PCR reaction high throughput) ultra-rapid cycling (30 minutes to 2 hours) highly sequence-specific

1. Why Real-time PCR ?

Disadvantages of real-time PCR

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

2. Theory of real-time PCR

Linear ground phase: 2. Theory of Real-time PCR 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 CT represents the starting copy no. in the original template PCR can be broken into 4 major phases

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 standard deviation of the baseline)

2. Theory of Real-time PCR

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

2. Theory of Real-time PCR

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

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

The Amplification Plot contains valuable information for the quantitative measurement of DNA or RNA. 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 the amplification 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 5 nuclease assay.

What is Rn?

2. Theory of Real-time PCR

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 the magnitude of the signal generated by the PCR 4. DRn is plotted against cycle numbers to produce the amplification curves and gives the CT value

Sample
Threshold

+Rn Rn

Rn

CT
cycle number 0
10 20

No Template Control 30 40

-Rn

3.Types of real-time PCR quantification a: Detection b: Calculation c: Normalization (in part4 )

Detection chemistries
Four common ways: 1.DNA binding dyes E.g. SYBR Green 2.Hydrolysis probes TaqMan 3.Hybridisation probes E.g. Light cycler 4.Hairpin probes Molecular beacons

3. PCR Quantification

SYBR Green (double-stranded DNA binding dye)

3. PCR Quantification

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 next heating cycle, the dye molecules are released and the fluorescence signal falls.

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

3. PCR Quantification

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 melting point curve determination

* longer amplicons create a stronger signal, amplicons should be 100 to 200 bp in size

3. PCR Quantification

When to choose SYBR Green

* Assays that do not require specificity of probe based 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 NOT to choose SYBR Green

* Allelic discrimination assays * Multiplex reactions * Amplification of rare transcripts * Low level pathogen detection from environmental samples or limited samples

The TaqMan 5 exonuclease assay

FRET = Frster/fluorescence resonance energy transfer & DNA Polymerase 5' exonuclease activity

3. PCR Quantification

Mocellin et al. Trends Mol Med 2003 (www)

FRET

3. PCR Quantification

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

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 emission
wavelength 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 the

nascent 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 longer

Human Molecular Genetics 2. NCBI Books (www)

in close proximity to the quencher, the resulting increase in reporter emission intensity is easily detected. 3. PCR Quantification

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 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.

Light cycler

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 light from 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.

3. PCR Quantification

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

Comparing three different fluorescence-monitoring systems

for DNA amplification.
Wittwer, 1997 (www)

3. PCR Quantification

Depends on the accumulated amplification product

Depends on the 5exonuclease activity of the polymerase

Depends on the independent hybridization of adjacent donar and acceptor probes

Molecular Beacons
1. Molecular beacons are the simplest hairpin probe flanked by 2 inverted repeats. 2. Reporter and quencher dyes are attached to each end of the molecule, causing a reduction in fluorescence emission in hairpin formation 3. When bound to the target, the quencher and reporter are separated, allowing reporter emission.
Thermodynamic stability: Probe-target helix > hairpin structure > mis-matched probe target helix

3. PCR Quantification

Mocellin et al. Trends Mol Med 2003 (www)

3.Types of real-time PCR quantification a: Detection b: Calculation c: Normalization (in part4 )

3. Types of real-time PCR quantification

* Absolute quantification
* Relative quantification (relative fold change) i. Relative standard method ii. Comparative CT (2 -CT) method

3. PCR Quantification

ASHI Quarterly

3. PCR Quantification

3. PCR Quantification

Absolute quantification
This method assumes all standards and samples have approximately equal amplification efficiencies More labour-intensive because of the necessity to create reliable standards for quantification include these standards in every PCR not possible to use DNA as a standard for absolute quantitation of RNA because there is no control for the efficiency of the reverse transcription Accurate determination of total RNA concentration is particularly important quantification by OD measurement faces problem of DNA contamination or inaccurate results from the spectrophotometer RNA constituting on average only 50-80% of the purified nucleic acid Additional step of DNase removal should be carried out prior to any RT step 3. PCR Quantification

3. Types of real-time PCR quantification

* 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 of an internal control RNA. relative quantification does not require standards with known concentrations

i. Relative standard method ii. Comparative CT (2 -CT) method

3. PCR Quantification

Relative standard curve method

1. Construct a relative standard curve
3. Divide the amount of c-myc by the amount of GAPDH to determine the normalized amount of c-myc (c-mycN).

2. Calculate the input amount by entering the following formula in an adjacent cell: = 10^ [cell containing log input amount] 3. PCR Quantification

Types of real-time PCR quantification

* Absolute quantification * Relative quantification (relative fold change vs calibrator) i. Relative standard curve method ii. Comparative CT (2 -CT) method

3. PCR Quantification

Validation experiment for comparative CT method

ABI-7700 User Bulletin #2

3. PCR Quantification

Comparative CT method-I

ABI-7700 User Bulletin #2

3. PCR Quantification

Comparative CT method - II

ABI-7700 User Bulletin #2

3. PCR Quantification

3. PCR Quantification

control

ref control

Ct = target - ref

Ct = 9.70
target control av =29.63

av =19.93

experiment

target treated ref treated

Ct = target - ref Ct = -1.7

av =18.03 av =19.80

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

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

2702

4. Housekeeping Gene for Normalization

None of the identified reference for data normalization are ideal.

1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 2. b -actin 3. Ribosomal RNA (rRNA): 28S, 18S

Housekeeping Gene for Normalization

GAPDH

4.

Housekeeping Gene for Normalization

(Bustin, 2000)

4. Housekeeping Gene for Normalization

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 (Mansur et 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 (Spanakis 1993),dexamethasone (Oikarinen et al. 1991) and carbon tetrachloride (Goldsworthy et al. 1993). Insulin stimulates GAPDH transcription (Rolland et al 1995, Barroso et al. 1999) calcium ionophore A23187 induces GAPDH transcription Growth hormone (Freyschuss et al. 1994), vitamin D (Desprez et al. 1992), oxidative stress (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 been shown to activate its transcription retinoic acid (Barroso et al. 1999) downregulate GAPDH transcription in the gut and in adipocytes, 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.

b-actin concentrations vary widely in response to

experimental manipulation in human breast epithelial cells (Spanakis 1993) in various porcine tissues (Foss et al. 1998) canine myocardium (Carlyle et al. 1996) the presence of pseudogenes interferes with the interpretation of results (Dirnhofer et al. 1995, Raff et al. 1997, Mutimer et al. 1998) primers commonly used for detecting -actin mRNA amplify DNA (Dakhama et al. 1996).

4.

Housekeeping Gene for Normalization

Common normalizing housekeeping gene

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) b -actin

Ribosomal RNA (rRNA)

28S, 18S
Varying ratios of rRNA to mRNA have been reported (Solanas et al.,2001) Use random hexamer instead of oligo dT in the RT step

4.

Housekeeping Gene for Normalization

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 junctions in cDNA to avoid genomic DNA being amplified
ABI Primer Express Software Tutorial (www)

4.

Housekeeping Gene for Normalization

Optimization of primers (Dissociation curve)

4.

Housekeeping Gene for Normalization

FINAL REMARKS: Doing real-time RT-PCR is easy, but making real time RT-PCR data a meaningful figure of qPCR mRNA expression profiles should have some more careful considerations such as:

Different detection chemistry fit different purposes Make a dilution of a template, either sDNA, sRNA or total RNA for a standard curve Correlation coefficient of the standard curve > 0.99? Normalization of samples obtained experiments can be carried out against 2 or 3 housekeeping genes whose expression has been shown to be unaffected by experimental conditions.

Good luck with your experiments and with your career!

4. Housekeeping Gene for Normalization

References
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3.

4.

Wong, M.L. and Medrano J.F. 2005 Real-time PCR for mRNA quantitation. Biotechniques 39(1): 75-85. Bustin, S.A. 2002 Quantification of mRNA using realtime reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23-39. Bustin, S.A. 2000 Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169-193. Websites from ABI, MJ companies, etc.