real-time polymerase chain reaction
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Real-time polymerase chain reaction
Presented by: Farhad Jahanfar
Introduction to PCR
PCR was invented in 1984 by ( Kary mullis ) & he
received the Nobel Prize in chemistry in 1993, for
his invention.(1)
It revolutionized biological methods specially in
molecular cloning in a way that it has
became an inseparable & irreplaceable part of
molecular investigations.
2
What you need for PCR• Together in a reaction tube on;
– Sample (+/- target DNA)
– Primers for the specific detection
– Nucleotides (dNTPs)
– Enzyme (taq polymerase, pfu or...)
– Buffer
– MgCl2
– Water
– Addetives( optional)Ficoll400 and tartrazine omit the need for gel loading buffer in both agarose and PAGE.
– Extras(enhancers)
Extraction Methods1) Organic (phenol/chloroform)
2) Non-Organic (Proteinase K and Salting out)
3) Chelex: styrene di-vinylbenzene copolymer containing paired imino –di-acetate ions
4) FTA Paper
5) Boiling
• Note: The method utilized may be sample dependant, techniquedependant, or analyst preference
DNA template troubleshooting
• DNA Template• Usually,100ng is
sufficient for a good PCR product (1 ngr-1 µgr).
• Amount of DNA present▫ Less DNA means more cycles, high means false
priming and • Complexity of DNA
▫ Ex. plasmid vs. whole genome• Purity
▫ Interfering factors, eg. enzymes, salts▫ If salting out was used in Extraction may
contamination by salts.• Degradation
▫ PCR more forgiving of degraded DNA• Contamination: urea, SDS, Na acetate
▫ Addition organic extractions, ethanol precipitation orPAGE Vs. agarose purification will decrease suchcontamination.
• Presence of “poisons”▫ Eg. EDTA which scavenges Mg 2+
Primer troubleshooting
• Age
• Number of freeze-thaws
• Contamination
• Amount
– Can vary over a wide range (50X)
– 100-500 nM typical( 0.1 -1 μm each primer)
– Too low: low amplification
– Too high: low amplification or unspecific bands, primer dimers
– Comment; use primers without complementary specially in 3’end.
– For short DNA fragments (100bp) may need more primers, ex;>1
μm of each primers
Buffer Considerations
• Most reaction buffers consist of a buffering agent, most
often a Tris-based buffer, and salt, commonly KCl.
• The buffer: regulates the pH of the reaction (affects the
DNA polymerase activity and fidelity).
• Modest concentrations of KCl will increase DNA
polymerase activity, by 50–60% over activities in the
absence of KCl; 50mM KCl is considered optimal.
•
Buffer Considerations• DNA Polymerase contains native Taq DNA polymerase in a
proprietary formulation. It is supplied with 5X GreenGoTaq® Reaction Buffer and 5X Colorless GoTaq.
5X Green GoTaq® Reaction Buffer: contains 2 dyes (blueand yellow) that separate during electrophoresis tomonitor migration progress and also contains a compoundthat increases the density of the sample to sink into thewell of the agarose gel, allowing reactions to be directlyloaded onto an agarose gel, No the need for loading dye.
The blue dye comigrates at the same rate as a 3–5kb DNAfragment in a 1% agarose gel.
The yellow dye migrates at a rate faster in a 1% agarosegel.
Buffer troubleshooting• Buffer
• To avoid contamination must aliquot insmall cap-tubes (avoid nucleasecontamination).
• Using high quality reagents.• Must match to polymerase.• Typically contain KCl and Tris.• Can vary over a slight range:
▫ Not much difference in range from 0.8 X to 2.0 X
▫ With or without dye.▫ Note: Primer efficiency reduced outside
this range.
Mg2+ concentration•Magnesium is a required cofactor for Taq DNA
polymerases, and Mg2+concentration is a crucial
factor that can affect amplification success.
• Template DNA concentration, chelating agents
present in the sample (e.g., EDTA or citrate),
dNTPs concentration and the presence of
proteins can all affect the amount of free Mg2+ in
the reaction.
•
•Figure: different amounts of free Mg2+, TaqDNA polymerase is inactive
Mg2+ concentration Excess free Mg2+, reduces enzyme fidelity and may
increase the level of nonspecific amplification.
So, empirically determine the optimal magnesiumconcentration for each target.
The effect of Mg2+ concentration and the optimalconcentration range can vary with the particular DNApolymerase. For example, the performance of Pfu DNApolymerase seems to be less dependent on Mg2+concentration.
Optimization is required, the optimal concentration isusually in the range of 2–6mM.
Mg2+ concentration• Many DNA polymerases are supplied with a Mg-free
reaction buffer and a tube of 25mM MgCl2 so that youcan adjust the Mg2+ concentration to the level that isoptimal for each reaction.
• Comment: Before assembling the reactions, be sure tothaw the magnesium solution completely prior to useand vortex the magnesium solution for several seconds(to obtain a uniform solution) before pipetting.
• .
• MgCl2 • Mg2+ is an essential cofactor of DNA polymerase and need for dsDNA establishment.
• Amount can vary:
– 0.5 to 3.5 μm (1-4μm) suggested
– Too low: Taq won’t work
– Too high: mispriming, PCR fidelitydecreasing, un-specific PCRproducts ( ladder formation orsmear)
dNTPs troubleshooting
• Nucleotides20-400 μM (10-200 each)works well
▫ - 200mM of each is enough to synthesize 12.5mg of DNA.
▫ Too much: can lead to mispriming and errors if >200mM each increase the error rate of polymerase.
▫ Too much: can scavenge Mg2+
▫ Too low: faint productsAgeNumber of freeze-thawsDilute in buffer (eg. 10mM Tris pH 7.4 -7.5 to
prevent acid hydrolysis)ContaminationFor long fragments need higher
dNTPsHigh change in dNTPs amount need
adjust of Mg2+
Enzyme Concentration
using 1–2.5 units of Taq DNA polymerase in a 50μl
amplification reaction, is recommended.
In most cases, an excess of enzyme, will not significantly
increase product yield.
In fact, increased amounts of enzyme increase the
likelihood of generating artifacts associated with the
intrinsic 5′→3′ exonuclease activity of Taq DNA
polymerase, resulting in smeared bands in an agarose gel.
Note: Pipetting errors are a frequent cause of excessive
enzyme levels.
• Taq Polymerase• Error rate 2x10-4N/C
• Thermostable! Activity declines with time at 95₀C(Hot start)
• Matches buffer• Age• Contamination• Concentration: Typically 0.5 to 1.0 U/for
a 25 μl reaction.
• If low; incompleteelongation(defective PCR products)
• If high: un specific bands and smearformation in gel.
• Note: usually adding 2.5 U/reactionMAY INCREASE but up to the point.
A Typical PCR Reaction
Sterile Water 38.0 ul
10X PCR Buffer 5.0 ul
MgCl2 (50mM) 2.5 ul
dNTP’s (10mM each) 1.0 ul
PrimerFWD (25 pmol/ul) 1.0 ul
PrimerREV 1.0 ul
DNA Polymerase 0.5 ul
DNA Template 1.0 ul
Total Volume 50.0 ul
Cycling• Cycling parameters: each step requires a minimal
amount of time to be effective, while too much timecan be both wasteful and deleterious to polymerase.
• Annealing step: Primers with low GC% needtemperature lower than 55°C.
• Extention step: temperature is usually 72°C, 1 min/kbproduct length is sufficient.
• Ramp time: the time it takes to change from onetemperature to another.
Choosing cycling parameters• Usual program
• 30 cycles: 30 sec 94ºC denaturation
• ---------------------------------------------------------------------------------------------
• 30 sec 55ºC (GC content≤50%) or annealing
• 60 ºC (GC content>50%)
• ------------------------------------------------------------------------------------------------
• 72ºC extension
.
Cycling is dependent upon
The sequence and length of the template DNA,
The sequence and complexity of primers,
The ramp times of the thermal cycler used,
The number of cycles depend on both the efficacy of reaction
and the amount of template DNA.
Note: Greater cycles No. (>40) can reduce the Taq efficacy and
increase the nonspecific bands and deplete substrate.
PCR Cycling Parameters
• Denaturation Step Must balance DNA denaturation with Taq damage.
95₀C for 30 - 60s typically is enough to denature DNA.
Even 92 ₀ C for 1s can be enough.
Taq loses activity at high temps:▫ Half-life at 95 ₀ C: 40 min
▫ Half-life at 97.5 ₀ C: 5 min
PCR Cycling Parameters
• Annealing Step
• Most critical step
• Calculate based on Tm.
Comment: Using an annealing temperature slightlyhigher than the primer Tm will increase annealingstringency and can minimize nonspecific primerannealing decrease the amount of undesired productssynthesized
▫ Often does not give expected results• Trial-and-Error
▫ Almost always must be done anyway
▫ Too hot: no products
▫ Too low: non-specific products
Comment: Gradient thermocyclers are very useful
• Typically only 20s needed for primers to anneal
PCR Cycling Parameters• Extension Step
• Temperature typically 72 ₀ C
– Reaction will also work well at 65₀ C or other temps
• Time (in minutes) roughly equal tosize of the largest product in kb
– Polymerase runs at 60bp/sunder optimum conditions
• Final “long” extension step mostlyunnecessary
PCR Cycling Parameters
• Number of Cycles • Source of DNA molecules:– >100,000: 25-30 cycles
– >10,000: 30-35 “
– >1,000: 35-40 “
– <50: 20-30 fb. nested PCR
• Do not run more than 40 cycles.– Virtually no gain
– Extremely high chance of non-specific products
• Best optimized by trial-and-error
Comment: If nonspecific amplification products accumulate, diluting the
products of the first reaction and performing a 2th amplification with the same
primers or primers that anneal to sequences within the desired PCR product
(nested primers).
Basic Experimental Design
• A well-designedexperiment can keep youfrom ever getting intotrouble!
Basic Experimental Design Comments
• Main point: Always use CONTROLS
• Positive control– So you’ll know what a
successful result looks like.
• Negative control– Lets you know if you have
contamination.
Experimental Design: Controls
No positive or negative controls…What does this result mean??
Only a positive control…How do we know the result isn’t due tocontamination?
Both positive and negative controls…Results can be interpreted withconfidence.
U
U +
U - +
Basic Experimental Design Comments
If no band , 1th add 10 cycles
Dilute 1th PCR product and re PCR
Increase the denaturation temperature
Add enhancers
Using TAE in Electrophoresis Vs. TBS can delete
the smears.
Basic Experimental Design PCR Cycling Parameters
• “Odd” Protocols • Hot-Start PCR
– Taq is added latter
• Touchdown PCR
– Annealing temperature is progressively reduced.
Extras troubleshooting
•Higher yields can achieve by:• A) By using Noninonic detergents (Triton X-100) neutrialize
charges of ionic detergents often use in DNA extraction.
• B) By using Taq DNA Polymerase stabilizing activity with
enzyme stabilizing proteins (BSA or gelatin)
• C) By using Polymerase stabilizing solutes (betain)
• D) By using Enzyme stabilizing solvents (glycerol)
• E) By using Solubility Enhancing solvents (DMSO)
• F) By using Molecular crowding solvents (PEG)
• G) By using Polymerase salt preference( NH4SO4)
Cont’d Greater specificity is achieved by:
Lowering Tm of dsDNA (by Formamide); lowers meltingtemperatures (Tm) of DNAs linearly by 2.4-2.9 C/mole offormamide, depending on the (G+C) composition, helixconformation and state of hydration.
Destabilizing mismatched primer annealing (by PMPE* or Hotstart strategies)
Amplification of high GC components by:
by using betain*
Other PCR Reaction Components
• Extras=
• enhancers
Added by user:
Glycerol(5-20%), DMSO(1-10%)Stabilize Taq, decrease secondary structure
Probable help or hurt, depending on primers
Typically already in the Taq stock
Betaine (1-2 M)Useful for GC-rich templates
For long templates, higher pH isrecommended(9.0), the pH of Tris bufferdecreases at high temperatures, long-templatePCR requires more time at high temperatures andincreasing time at lower pH may cause somedepurination of the template resulting in reducedyield of specific product.
Cont’d
PEG 6000(5-15%)
Non ioning detergents
Foramide (1.25%-10%), DMSO (1–10%) or BSA (10-100 μg/ml)
which frequently helps, doesn’t hurt.
Note: Concentrations of DMSO greater than 10% and
formamide greater than 5% can inhibit Taq DNA
polymerase and presumably other DNA polymerases as
well.
Analyze the productelectrophoresis
• Agarose
Analyze the productelectrophoresis
• PAGE
Analyze the product Electrophoresis 10µl from each reaction on an agarose
and nondenaturing PAGE, for resolution of PCR products
between 100 to 1000bp.
Further staining by Ethidium bromide (Etbr) or SYBR (25
to 200) times more sensitive than Etbr, more convenient
to use, and permits optimization of 10 to 100 fold lower
starting template copy No. Silver staining in PAGE (
more sensitive)
What do mRNA levels tell us?
DNAmRNAprotein
• Reflect level of gene expression
• Information about cell response
• Protein production (not always)
quantitative mRNA/DNA analysis
Direct
-Northern blotting
-In situ hybridization
PCR amplification
-Regular RT-PCR
-Real time PCR
(Microarrays)
Nomenclature
RT-PCR = Reverse Transcriptase PCR
qReal time PCR = quantitative Real-Time PCR
RT-PCR
• Isolate RNA
• cDNA synthesis
• PCR reaction
Annealing of Downstream Primer to RNA
Reverse Transcription With AMV Reverse Transcriptase
RNA Copied From 3’ to 5’ into cDNA
Amplification of cDNA by PCR
Why isn´t this good enough?
What’s Wrong With
Agarose Gels?
* Low sensitivity
* Low resolution
* Non-automated
* Size-based discrimination only
* Results are not expressed as numbers
based on personal evaluation
• Ethidium bromide staining is not very quantitative
• End point analysis
Different concentrations give similar endpoint results!
Endpoint analysis
How does real-time PCR work?
To best understand what real-time PCR
is, let’s review how regular PCR
works...
The Polymerase Chain Reaction
How does PCR work??5’
5’
3’
3’
d.NTPs
Thermal Stable DNA Polymerase
Primers
Denaturation
Annealing
Add to Reaction Tube
The Polymerase Chain Reaction
How does PCR work??
Extension
5’ 3’
5’3’
Extension Continued
5’ 3’
5’3’
Taq
Taq
3’
5’3’
Taq
Taq
Repeat
The Polymerase Chain Reaction
How does PCR work??
5’3’
3’
3’
3’
5’3’3’
5’3’
3’Cycle 2
4 Copies
Cycle 3
8 Copies
3’
3’
5’3’
3’
5’3’
3’
5’3’
3’
5’3’
3’
5’3’3’
5’3’
3’
5’3’
3’
Imagining Real-Time
PCR
…So that’s how PCR is usually presented.
To understand real-time PCR, let’s imagine
ourselves in a PCR reaction tube at cycle
number 25…
Imagining Real-Time
PCR
What’s in our tube, at cycle number 25?
A soup of nucleotides, primers, template,
amplicons, enzyme, etc.
1,000,000 copies of the amplicon right now.
Imagining Real-Time
PCR
How did we get here?
What was it like last cycle, 24?
Almost exactly the same, except there were only 500,000 copies of the amplicon.
And the cycle before that, 23?
Almost the same, but only 250,000 copies of the amplicon.
And what about cycle 22?
Not a whole lot different. 125,000 copies of the amplicon.
Imagining Real-Time PCR
How did we get here?
If we were to graph the amount of DNA in our
tube, from the start until right now, at cycle
25, the graph would look like this:
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
0 5 10 15 20 25 30 35 40
Imagining Real-Time PCR
How did we get here?
So, right now we’re at cycle 25 in a soup with
1,000,000 copies of the target.
What’s it going to be like after the next cycle,
in cycle 26?
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
0 5 10 15 20 25 30 35 40
?
Imagining Real-Time PCR
So where are we going?
What’s it going to be like after the next cycle, in cycle 26?
Probably there will be 2,000,000 amplicons.
And cycle 27?
Maybe 4,000,000 amplicons.
And at cycle 200?
In theory, there would be 1,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000 amplicons…
Or 10^35 tonnes of DNA…
To put this in perspective, that would be equivalent to the weight of ten billion planets the size of Earth!!!!
Imagining Real-Time
PCR
So where are we going?
A clump of DNA the size of ten billion planets
won’t quite fit in our PCR tube anymore.
Realistically, at the chain reaction progresses,
it gets exponentially harder to find primers,
and nucleotides. And the polymerase is
wearing out.
So exponential growth does not go on
forever!
Imagining Real-Time PCR
So where are we going?
If we plot the amount of DNA in our tube
going forward from cycle 25, we see that it
actually looks like this:
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
0 5 10 15 20 25 30 35 40
MeasuringQuantities
How can all this be used to measure DNA
quantities??
What if YOU started with FOUR times as much
DNA template as I did?
I have 1,000,000 copies at cycle 25.
You have 4,000,000 copies!
So… You had 2,000,000 copies at cycle 24.
And… You had 1,000,000 copies at cycle 23.
Imagining Real-Time
PCR
MeasuringQuantities
So… if YOU started with FOUR times as much
DNA template as I did…
Then you’d reach 1,000,000 copies exactly
TWO cycles earlier than I would!
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
0 5 10 15 20 25 30 35 40
MeasuringQuantities
What if YOU started with EIGHT times LESS DNA template
than I did?
You’d only have 125,000 copies right now at cycle 25…
…and you’ll have 250,000 at 26, 500,000 at 27, and by cycle
28 you’ll have caught up with 1,000,000 copies!
So… you’d reach 1,000,000 copies exactly THREE cycles
later than I would!
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
0 5 10 15 20 25 30 35 40
MeasuringQuantities
We describe the position of the lines with a value that
represents the cycle number where the trace crosses
an arbitrary threshold.
This is called the “Ct Value”.
Ct values are directly related to the starting quantity of
DNA, by way of the formula:
Quantity = 2^Ct
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
0 5 10 15 20 25 30 35 40
23 25 28
Ct Values:
67
Imagining Real-Time
PCR
MeasuringQuantities
There’s a DIRECT relationship between the
starting amount of DNA, and the cycle
number that you’ll reach an arbitrary
number of DNA copies (Ct value).
DNA amount = 2 ^ Cycle Number
C o p y N u m b e r v s. C t - Sta n d a r d C u r v e
y = -3 . 3 1 9 2 x + 3 9 . 7 7 2
R2 = 0 .9 9 6 7
0
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
0 1 2 3 4 5 6 7 8 9 1 0 1 1
Lo g o f co p y n u m b er (1 0 n )
Ct
Real-time PCR advantages
* not influenced by non-specific amplification
* amplification can be monitored real-time
* no post-PCR processing of products(high throughput, low contamination risk)
* requirement of 1000-fold less RNA than conventional assays(3 picogram = one genome equivalent)
* most specific, sensitive and reproducible
Real-time PCR disadvantages
* setting up requires high technical skill and support
* high equipment cost
* Runs are more expensive than conventional PCR
* DNA contamination (in mRNA analysis)
Cycle Threshold
* cycle threshold or the CT value is the cycle at which a significant increase in DRn is first detected
* it is the parameter used for quantitation
* CT value of 40 or more means no amplification and cannot be included in the calculations
Data analysis
PCR phases in linear view
Cycle #
[DNA]
PCR phases
Exponential◦ If 100% efficiency – exact doubling of products.
Specific and precise
Linear◦ High variability. Reaction components are being
consumed and PCR products are starting to degrade.
Plateau◦ End-point analysis. The reaction has stopped and if left
for long – degradation of PCR products.
72
more…
Exponential
Linear
Plateau
PCR buffer
dNTP Mix
Thermostable DNA polymerase
Template
DDW
…
73
74
Gene of interest
Mutation Detection
Allelic discrimination
Gene expression ( mRNA)
Microbial agents detection
Quantification
…Disease
General rules for primer design
-- Specificity and cross homology
Specificity
Determined primarily by primer length as well as sequence
The adequacy of primer specificity is dependent on the nature of the
template used in the PCR reaction.
Cross homology
Cross homology may become a problem when PCR template is genomic
DNA or consists of mixed gene fragments.
Primers containing highly repetitive sequence are prone to generate non-
specific amplicons when amplifying genomic DNA.
Avoid non-specific amplification
BLASTing PCR primers against NCBI non-redundant sequence database
is a common way to avoid designing primers that may amplify non-
targeted homologous regions.
Primers spanning intron-exon boundaries to avoid non-specific
amplification of gDNA due to cDNA contamination.
Primers spanning exon-exon boundaries to avoid non-specific amplification
cDNA due to gDNA contamination.
General rules for primer design
-- Primer and amplicon length
Primer length determines the specificity and
significantly affect its annealing to the template
Too short -- low specificity, resulting in non-specific
amplification
Too long -- decrease the template-binding efficiency at
normal annealing temperature due to the higher probability
of forming secondary structures such as hairpins.
Optimal primer length
18-24 bp for general applications
30-35 bp for multiplex PCR
Optimal amplicon size
300-1000 bp for general application, avoid > 3 kb
50-150 bp for real-time PCR, avoid > 400 bp
General rules for primer design
-- Melting temperature (Tm)
Tm is the temperature at which 50% of the DNA duplex
dissociates to become single stranded
Determined by primer length, base composition and concentration.
Also affected by the salt concentration of the PCR reaction mix
Working approximation: Tm=2(A+T)+4(G+C) (suitable only for 18mer
or shorter).
Optimal melting temperature
52°C-- 60°C
Tm above 65°C should be generally avoided because of the potential for
secondary annealing.
Higher Tm (75°C-- 80°C) is recommended for amplifying high GC
content targets.
Primer pair Tm mismatch
Significant primer pair Tm mismatch can lead to poor amplification
Desirable Tm difference < 5°C between the primer pair
General rules for primer design
-- Annealing temperatures and other considerations
Ta (Annealing temperature) vs. Tm
Ta is determined by the Tm of both primers and amplicons:
optimal Ta=0.3 x Tm(primer)+0.7 x Tm(product)-25
General rule: Ta is 5°C lower than Tm
Higher Ta enhances specific amplification but may lower yields
Crucial in detecting polymorphisms
Primer location on template Dictated by the purpose of the experiment
For detection purpose, section towards 3’ end may be preferred.
When using composite primers Initial calculations and considerations should emphasize on the template-
specific part of the primers
Consider nested PCR
General rules for primer design
-- GC content; repeats and runs
Primer G/C content
Optimal G/C content: 45-55%
Common G/C content range: 40-60%
Runs (single base stretches)
Long runs increases mis-priming (non-specific annealing)
potential
The maximum acceptable number of runs is 4 bp
Repeats (consecutive di-nucleotide)
Repeats increases mis-priming potential
The maximum acceptable number of repeats is 4 di-
nucleotide
General rules for primer design
-- Primer secondary structures
Hairpins
Formed via intra-molecular interactions
Negatively affect primer-template binding, leading to poor or no
amplification
Acceptable ΔG (free energy required to break the structure): >-2
kcal/mol for 3’end hairpin; >-3 kcal/mol for internal hairpin;
Self-Dimer (homodimer)
Formed by inter-molecular interactions between the two same primers
Acceptable ΔG: >-5 kcal/mol for 3’end self-dimer; >-6 kcal/mol for
internal self-dimer;
Cross-Dimer (heterodimer)
Formed by inter-molecular interactions between the sense and antisense
primers
Acceptable ΔG: >-5 kcal/mol for 3’end cross-dimer; >-6 kcal/mol for
internal cross-dimer;
General rules for primer design
-- GC clamp and max 3’ end stability
GC clamp
Refers to the presence of G or C within the last 4 bases from
the 3’ end of primers
Essential for preventing mis-priming and enhancing specific
primer-template binding
Avoid >3 G’s or C’s near the 3’ end
Max 3’end stability
Refers to the maximum ΔG of the 5 bases from the 3’end of
primers.
While higher 3’end stability improves priming efficiency, too
higher stability could negatively affect specificity because of
3’-terminal partial hybridization induced non-specific
extension.
Avoid ΔG < -9.
Primer Design Considerations
Consideration Comment
Primer Length 18-30 bases
Primer Melting Temperature (Tm) 55°-72°C
Primer Annealing Temperature (Ta) ~5°C < the lowest Tm of the of primers
Tm difference between forward and reverse primers ≤ 5°C
Max 3′ Stability ∆G value for five bases from 3′ end
Percentage GC content 40-60%
No Secondary StructuresIdentify primer pairs which do not assume
secondary structure
No self-complementarity < 4 contiguous bases
No complementarity to other primer(s) < 4 contiguous bases
No long runs with the same base < 4 contiguous bases
Distance between two primers on target sequence < 2000 bases apart
Plateau Effect accumulation of product ≤0.3 to 1 pmol
83
Tool name URL
CODEHOP http://blocks.fhcrc.org/codehop.html
Gene Fisher http://bibiserv.techfak.uni-bielefeld.de/genefisher/
DoPrimer http://doprimer.interactiva.de/
Primer3 http://frodo.wi.mit.edu/primer3/
Primer Selection Http://alces.med.umn.edu/rawprimer.html
Web Primer http://genome.www2.stanford.edu/cgi.bin/SGD/web.primer
PCR designer http://cedar.genetics.ston.ac.uk/public_html/primer.html
Primo pro 3.4 http://www.changbioscience.com/primo.html
Primo Degenerate
3.4
http://www.changbioscience.com/primo/primod.html
PCR Primer Design http://pga.mgh.harvard.edu/serviet/org.mgh.proteome.primer
The Primer
Generator
http://www.med.jhu.edu/medcenter/primer/primer.cgi
EPRIMERS http://bioweb.pasteur.fr/seqanal/interfaces/eprimer3.html
PRIMO http://bioweb.pasteur.fr/seqanal/interfaces/eprimo.html3
PrimerQuest http://www.idtdna.com/biotools/primer_quest/primer_quest.asp
MethPrimer http://itsa.uscf/~uralab/methprimer/index1.html
Rawprimer http://alces.med.umn.edu/rawprimer.html
MEDUSA http://www.cgr.ki.se/cgr/MEDUSA/
The Primer Prim’er
Project
http://www.nmr.cabm.rutgers.edu/bioinformatics/primer_primer_proj
ect/primer.html
GAP http://promoter.ics.uci.edu/primers/
84
Description Software name
Analyses a template DNA sequence and chooses primer pairs for PCR and
primers for DNA sequencing
Primerselect
DANASIS Max is a fully integrated program that includes a wide range of
standard sequence analysis features.
DANSIS Max
Primer design for windows and power macintosh. Primer Primer 5
Comprehensive primer design for windows and Power Macintosh. Primer Primer:
Comprehensive analysis of individual primers and primer pairs. NetPrimer
For fast, effective design of specific oligos or PCR primer pairs for microarrays. Array Designer 2
Design molecular beacons and TaqMan probes for robust amplification and
fluorescence in real time PCR.
AlleleID 7
Primer design for DNA-arrays/chips. GenomePRIDE 1.0
Software for Microsoft Windows has specific. Ready-to-use template for many
PCR and sequencing applications; standard and long PCR inverse PCR.
Degenerate PCR directly on amino acid sequence. Multiplex PCR.
Fast PCR
Primer Analysis Software for Mac and Windows. OLIGO 7
Will find optimal primers in target regions of DNA or protein molecules, amplify
leatures in molecules, or create products of a specified length.
Primer Designer 4
Software for primer design. GPRIME
Genome Oligo Designer is a Software for automatic large scale design of optimal
oligonucleotide probes for microarray experiments.
Sarani Gold
Primer and template design and analysis. PCR Help
Genorama Chip Design Software is a complete set of programs required for
genotyping chip design.The programs can also be bought separately.
Genorama chip Design
Software
The Primer Designer features a powerful, yet extremely simple, real-time interface
to allow the rapid identification of theoretical ideal primers for your PCR
reactions.
Primer Designer
Automatic design tools for PCR. Sequencing or hybridization probes, degenerate
primer design, restriction, Nested/Multiplex primer design, restriction enzyme
analysis and more.
Primer Primer
DOS-program to choose primer for PCR or oligonucleotide probes. PreimerDesign
85
86
87
88
Mature RNA
Primary transcript
89
Exon 1 Exon 3Exon 2
This primer spans the exon 2- exon3 junction
Exon 1 Exon 2 Exon 3
90
91
Imagining Real-Time
PCR
What’s in our tube, at cycle number 25?
A soup of nucleotides, primers, template,
amplicons, enzyme, etc.
1,000,000 copies of the amplicon right now.
Intercalating Dyes ( SYBR GreenІ )
Hydrolysis probes ( TaqMan )
Molecular Beacons
FRET probes
Scorpions
AND …
General methods for Real time PCR detection
93
5’
5’
3’
3’
d.NTPs
Thermal Stable DNA Polymerase
Primers Add Master Mix and Sample
Denaturation
Annealing
Reaction Tube
SYBR GreenІ
Intercalation Dyes
Taq ID
l
Extension
5’ 3’
5’3’
Extension ContinuedApply ExcitationWavelength
5’ 3’
5’3’
Taq
Taq
3’
5’3’
Taq
Taq
Repeat
SYBR GreenІ
ID ID
ID IDID
ID ID ID
ID ID
l l l
ll
SYBR GreenI Primer Characteristics
Many of the guidelines for primer design are also
applicable to SYBR Green primer design.
Create amplicon length of 200-300 bp.
Avoid primer-dimers!!!
96
R
Q
RQ
TaqManTM probe
After cleveage
Before cleveage
TaqManTM
5’
5’
3’
3’
d.NTPs
Thermal Stable DNA Polymerase
Primers Add Master Mix and Sample
Denaturation
Annealing
Reaction Tube
Taq
l
R Q
Probe
R Q
5’ 3’
TaqManTM
RQ
Extension Step
5’ 3’ 1. Strand DisplacementTaq
Q
R
5’ 3’
Q Taq
R
5’ 2. Cleavage
3. PolymerizationComplete
5’ 3’
TaqR
5’
4. Detection5’ 3’
TaqR
5’
l
R
TaqMan Design characteristics-A
Many of the guidelines for primer design are also
applicable to TaqMan® probe design.
The probe melting temperature in general should be ~10؛C
higher than the forward or reverse primer.
The probe should be as close to the forward OR reverse
primers as possible, within 10 base pairs of the primer
that anneals to the same strand as the probe without
any overlaps
100
TaqMan Design characteristics-B
The amplicon size is usually in the range of 60–150.
Do not put G at the 5’ end of the probe as this will quench
reporter fluorescence.
In general, probes with more G than C bases will often
produce reduced normalized fluorescence values.
101
102
Molecular beacons
Molecular beacons
Reporter
Non-fluorescent Quencher
Amplicon
A
B
C
FRET
Excitation
ANNEALING
FITC
Red 640
P Phosphate
FRET Emission
P
Excitation
Amplicon
FRET Design characteristics
Many of the guidelines for primer design are also applicable
to FRET probe design.
Amplicon should be between 100-200.
Place probes far away from the primer on the same strand.
Length of 25 to 35 nucleotides.
TM 5 to 8°C above primer TM.
No Gs should be located in the target strand between the
annealed probes.
105
The Basic of Real time PCR
Baseline – The baseline phase contains all the amplification that is below the level of
detection of the real time instrument.
Threshold – where the threshold and the amplification plot intersect defines CT. Can be set manually/automatically
CT – (cycle threshold) the cycle number where the fluorescence passes the threshold
DRn – (Rn-baseline)
NTC – no template control
RT control
Melting Curve
Log
flu
ore
scen
ce (
Rn
)
NTC Controls
NTC’s- Negative Control Rxn with Primers but without Template Examine Amp Plots to determine whether or not you have any amplification products.
The presence of an amplication product is indicative of contamination
Sources of contamination: water, primers, SYBR Green Master Mix, Pipets, etc
RT control
• Genomic DNA contamination
• Primer design (Exon-exon junction)
Melt Curve Analysis
Melt Curve used to Assess Specificity of Rxn Do you have a single amplification peak or multiple amplification peaks?
Single Peak= Single Product
Multiple Peaks= Multiple Products
What is the peak melt temperature and how does this correlate with the expected melt peak temperature?
To further verify that you have amplified correct product can sequence amplification product
Output data: Melt curve*
Relative fluorescence units
Gradual temperature-dependent fluorescencequenching
Rapid decrease in fluorescence caused by denaturation of dsDNA (PCR product)
Negative first derivative offluorescence/temperature
Melt peak (85.5º C)
Identification of multiple PCR products using a melt curve
product 2(Tm 86.9º C)
Under identical solvent conditions, Tm is determined by G/C content and length of dsDNA
primer dimer(Tm 77.0º C) product 1
(Tm 85.5º C)
Reverse Transcription
Total RNA 5 g
random hexamers (50 ng/l) 3 l
10 mM dNTP mix 1 l
DEPC H2O to 10 l
10x RT buffer 2 l
25 mM MgCl2 4 l
0.1 M DTT 2 l
RNAaseOUT 1 l
Incubate the samples at 65C for 5 min and then on ice for at least 1 min.
Add 1 µl (50 units) of SuperScript II RT to each tube, mix and incubate at 25C for 10 min.
Incubate the tubes at 42C for 50 min, heat inactivate at 70C for 15 min, and then chill on ice.
25 l SYBR Green Mix (2x)
0.5 l cDNA
2 l primer pair mix (5 pmol/l each primer)
22.5 l H2O
OR
12.5 l SYBR Green Mix (2x)
0.2 l cDNA
1 l primer pair mix (5 pmol/l each primer)
11.3 l H2O
Tth DNA Polymerase
Platinum® Taq DNA polymerase
GoTaq® Hot Start Polymerase
Hot Start II High-Fidelity DNA Polymerase
95C 10 min, 1 cycle 95 C 15 s -> 60 C 30 s -> 72 C 30 s, 40 cycles
Results interpretation
Following run evaluation
Valid positive and negative control
Specimen has a normal curve
Record the cycle threshold (Ct) values
If a sample has no cycle threshold values (0.00) it is negative
Determine if there are any suspect samples
Weak positives- Ct values >35
Results interpretation
Following run evaluation
Valid positive and negative control
Specimen has a normal curve
Record the cycle threshold (Ct) values
If a sample has no cycle threshold values (0.00) it is negative
Determine if there are any suspect samples
Weak positives- Ct values >35
* Absolute quantification* Relative quantification (relative fold change)
i. Relative standard method ii. Comparative CT (2 -DDCT) method
Types of real-time PCR quantification
118
STANDARD CURVE
METHOD
120
CYCLE NUMBER AMOUNT OF DNA
0 1
1 2
2 4
3 8
4 16
5 32
6 64
7 128
8 256
9 512
10 1,024
11 2,048
12 4,096
13 8,192
14 16,384
15 32,768
16 65,536
17 131,072
18 262,144
19 524,288
20 1,048,576
21 2,097,152
22 4,194,304
23 8,388,608
24 16,777,216
25 33,554,432
26 67,108,864
27 134,217,728
28 268,435,456
29 536,870,912
30 1,073,741,824
31 1,400,000,000
32 1,500,000,000
33 1,550,000,000
34 1,580,000,000
121
0
200000000
400000000
600000000
800000000
1000000000
1200000000
1400000000
1600000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBERA
MO
UN
T O
F D
NA
1
10
100
1000
10000
100000
1000000
10000000
100000000
1000000000
10000000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBER
AM
OU
NT
OF
DN
A
CYCLE NUMBER AMOUNT OF DNA
0 1
1 2
2 4
3 8
4 16
5 32
6 64
7 128
8 256
9 512
10 1,024
11 2,048
12 4,096
13 8,192
14 16,384
15 32,768
16 65,536
17 131,072
18 262,144
19 524,288
20 1,048,576
21 2,097,152
22 4,194,304
23 8,388,608
24 16,777,216
25 33,554,432
26 67,108,864
27 134,217,728
28 268,435,456
29 536,870,912
30 1,073,741,824
31 1,400,000,000
32 1,500,000,000
33 1,550,000,000
34 1,580,000,000
122
0
200000000
400000000
600000000
800000000
1000000000
1200000000
1400000000
1600000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBER
AM
OU
NT
OF
DN
A
0
200000000
400000000
600000000
800000000
1000000000
1200000000
1400000000
1600000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBER
AM
OU
NT
OF
DN
A
123
1
10
100
1000
10000
100000
1000000
10000000
100000000
1000000000
10000000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBER
AM
OU
NT
OF
DN
A
1
10
100
1000
10000
100000
1000000
10000000
100000000
1000000000
10000000000
0 5 10 15 20 25 30 35
PCR CYCLE NUMBER
AM
OU
NT
OF
DN
A
124
SERIES OF 10-FOLD DILUTIONS
threshold
Ct
125
126
127
15SERIES OF 10-FOLD DILUTIONS
threshold
• 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
Absolute quantification
Relative standard curve method1. Construct a relative
standard 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 by the amount of GAPDH to determine the
normalized amount of c-myc (c-mycN).
* Absolute quantification* Relative quantification
(relative fold change vs calibrator)i. Relative standard curve methodii. Comparative CT (2 -DDCT) method
Types of real-time PCR quantification
3. PCR Quantification
Quantification and Normalization
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
0 5 10 15 20 25 30 35 40
• First basic underlying principle: every cycle there is a doubling of product.
• Second basic principle: we do not need to know exact quantities of DNA, instead we will only deal with relative quantities.
• Third basic principle: we have to have not only a “target” gene but also a “normalizer” gene.
• Key formula:
– Quantity = 2 ^ (Cta
– Ctb
)
Housekeeping Gene for Normalization
Housekeeping Gene for Normalization
1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)2. b -actin3. Ribosomal RNA (rRNA): 28S, 18S
None of the identified reference for data normalization are ideal.
GAPDH
(Bustin, 2000)
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 inappropriateIt is a mystery why GAPDH continues to find favor as an internal standard.
4. Housekeeping Gene for Normalization
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”
•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
Comparative CT method-I
ABI-7700 User Bulletin #2
Comparative CT method - II
ABI-7700 User Bulletin #2
Relative quantification
Gapdh IL-2 delta delta delta RGE
Cont1 15 27 12 0 1 32
RA1 16 23 7 -5 32
Gapdh IL-2 delta delta delta RGE
Cont1 15 27 12 5 0.03125 32
RA1 16 23 7 0 1
10
Gapdh IL-2 delta delta delta RGE 32
Cont1 15 27 12 2 0.25
RA1 16 23 7 -3 8
0
Gapdh IL-2 delta delta deltaRGE
Cont1 15 27 12 12 0.00024414 1 32
RA1 16 23 7 7 0.0078125 32
targettreated
ref treated
targetcontrol
ref control
av =19.80
av =19.93
av =18.03
av =29.63
D Ct = 9.70
D Ct = -1.7
D Ct = target - ref
D Ct = target - ref
Difference = DCt-DCt= DDCt= (-1.7) -9.70= -11.40
control
experiment
Exercise: By 2 –∆∆CT, fold change=??? 2702
3. PCR Quantification
145
PFAFFL METHOD
– M.W. Pfaffl, Nucleic Acids
Research 2001 29:2002-2007
EFFECTS OF EFFICIENCY
146
147
AFTER 1 CYCLE100% = 2.00x90% = 1.90x80% = 1.80x70% = 1.70x
CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA
100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY
0 1 1 1 1
1 2 2 2 2
2 4 4 3 3
3 8 7 6 5
4 16 13 10 8
5 32 25 19 14
6 64 47 34 24
7 128 89 61 41
8 256 170 110 70
9 512 323 198 119
10 1,024 613 357 202
11 2,048 1,165 643 343
12 4,096 2,213 1,157 583
13 8,192 4,205 2,082 990
14 16,384 7,990 3,748 1,684
15 32,768 15,181 6,747 2,862
16 65,536 28,844 12,144 4,866
17 131,072 54,804 21,859 8,272
18 262,144 104,127 39,346 14,063
19 524,288 197,842 70,824 23,907
20 1,048,576 375,900 127,482 40,642
21 2,097,152 714,209 229,468 69,092
22 4,194,304 1,356,998 413,043 117,456
23 8,388,608 2,578,296 743,477 199,676
24 16,777,216 4,898,763 1,338,259 339,449
25 33,554,432 9,307,650 2,408,866 577,063
26 67,108,864 17,684,534 4,335,959 981,007
27 134,217,728 33,600,615 7,804,726 1,667,711
28 268,435,456 63,841,168 14,048,506 2,835,109
29 536,870,912 121,298,220 25,287,311 4,819,686
30 1,073,741,824 230,466,618 45,517,160 8,193,466
0
200,000,000
400,000,000
600,000,000
800,000,000
1,000,000,000
1,200,000,000
0 10 20 30
148
AFTER 1 CYCLE100% = 2.00x90% = 1.90x80% = 1.80x70% = 1.70x
AFTER N CYCLES:fold increase = (efficiency)n
CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA
100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY
0 1 1 1 1
1 2 2 2 2
2 4 4 3 3
3 8 7 6 5
4 16 13 10 8
5 32 25 19 14
6 64 47 34 24
7 128 89 61 41
8 256 170 110 70
9 512 323 198 119
10 1,024 613 357 202
11 2,048 1,165 643 343
12 4,096 2,213 1,157 583
13 8,192 4,205 2,082 990
14 16,384 7,990 3,748 1,684
15 32,768 15,181 6,747 2,862
16 65,536 28,844 12,144 4,866
17 131,072 54,804 21,859 8,272
18 262,144 104,127 39,346 14,063
19 524,288 197,842 70,824 23,907
20 1,048,576 375,900 127,482 40,642
21 2,097,152 714,209 229,468 69,092
22 4,194,304 1,356,998 413,043 117,456
23 8,388,608 2,578,296 743,477 199,676
24 16,777,216 4,898,763 1,338,259 339,449
25 33,554,432 9,307,650 2,408,866 577,063
26 67,108,864 17,684,534 4,335,959 981,007
27 134,217,728 33,600,615 7,804,726 1,667,711
28 268,435,456 63,841,168 14,048,506 2,835,109
29 536,870,912 121,298,220 25,287,311 4,819,686
30 1,073,741,824 230,466,618 45,517,160 8,193,466
0
200,000,000
400,000,000
600,000,000
800,000,000
1,000,000,000
1,200,000,000
0 10 20 30
149
0
200,000,000
400,000,000
600,000,000
800,000,000
1,000,000,000
1,200,000,000
0 10 20 30
PCR CYCLE NUMBER
AM
OU
NT
OF
DN
A
100% EFF
90% EFF
80% EFF
70% EFF
1
10
100
1,000
10,000
100,000
1,000,000
10,000,000
100,000,000
1,000,000,000
10,000,000,000
0 10 20 30
PCR CYCLE NUMBER
AM
OU
NT
OF
DN
A
100% EFF
90% EFF
80% EFF
70% EFF
CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA
100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY
0 1 1 1 1
1 2 2 2 2
2 4 4 3 3
3 8 7 6 5
4 16 13 10 8
5 32 25 19 14
6 64 47 34 24
7 128 89 61 41
8 256 170 110 70
9 512 323 198 119
10 1,024 613 357 202
11 2,048 1,165 643 343
12 4,096 2,213 1,157 583
13 8,192 4,205 2,082 990
14 16,384 7,990 3,748 1,684
15 32,768 15,181 6,747 2,862
16 65,536 28,844 12,144 4,866
17 131,072 54,804 21,859 8,272
18 262,144 104,127 39,346 14,063
19 524,288 197,842 70,824 23,907
20 1,048,576 375,900 127,482 40,642
21 2,097,152 714,209 229,468 69,092
22 4,194,304 1,356,998 413,043 117,456
23 8,388,608 2,578,296 743,477 199,676
24 16,777,216 4,898,763 1,338,259 339,449
25 33,554,432 9,307,650 2,408,866 577,063
26 67,108,864 17,684,534 4,335,959 981,007
27 134,217,728 33,600,615 7,804,726 1,667,711
28 268,435,456 63,841,168 14,048,506 2,835,109
29 536,870,912 121,298,220 25,287,311 4,819,686
30 1,073,741,824 230,466,618 45,517,160 8,193,466
0
200,000,000
400,000,000
600,000,000
800,000,000
1,000,000,000
1,200,000,000
0 10 20 30
150
1
10
100
1,000
10,000
100,000
1,000,000
10,000,000
100,000,000
1,000,000,000
10,000,000,000
0 10 20 30
PCR CYCLE NUMBER
AM
OU
NT
OF
DN
A
100% EFF
90% EFF
80% EFF
70% EFF
151
IL1-b con
IL1-b vit
RPLP0 vit
RPLP0 con
152
IL1-b con
IL1-b vit
AFTER N CYCLES: change = (efficiency)n
AFTER N CYCLES: ratio vit/con = (1.93)29.63-18.03 =1.9311.60 = 2053
av =18.03 av =29.63
IL1-beta
153
ratio = change in IL1-B = 2053/1.08 = 1901change in RPLP0
ratio = (Etarget )DCt target (control-treated)
(Eref )DCt ref (control-treated)
154
DDCt EFFICIENCY METHOD
APPROXIMATION METHOD
155
IL1-b con
IL1-b vit
RPLP0 vit
RPLP0 con
156
IL1-b vit
RPLP0 vit
IL1-b con
RPLP0 con
av =19.80
av =19.93
av =18.03
av =29.63
D Ct = 9.70
D Ct = -1.7
D Ct = target - ref
D Ct = target - ref
Difference = DCt-DCt= DDCt = 9.70-(-1.7)= 11.40
control
experiment
DDCt = 11.40 for IL1-beta
• 2 DDCt variant: assumes efficiency is 100% Fold change = 211.40 = 2702
• But our efficiency for IL1-beta is 93%
– Fold change = 1.9311.40 = 1800
• Pfaffl equation corrected for RPLP0 efficiency
– Fold change = 1901
157
• assumes
– minimal correction for the standard gene, or
– that standard and target have similar efficiencies
• 2 DDCt variant assumes efficiencies are both 100%
• approximation method, but need to validate that assumptions are reasonably correct - do dilution curves to check DCts don’t change
• The only extra information needed for the Pfaffl method is the reference gene efficiency, this is probably no more work than validating the approximation method
158
DDCt EFFICIENCY METHOD
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