Dr. Chaim Wachtel
Introduction to PCR and qPCR Part II:
PCR!!
qPCR technical workflow
Sampling
DNA Extraction
RNA Extraction
DNase treatment
ReverseTranscription
qPCR DataAnalysis
Primer design
Primer design – key to successful PCR
• Good primer design saves time and money• Advanced applications require even more
stringent primer design– Multiplex– Low abundance
Good primer (pair) properties
Primers should have• 18-24 bases• 40-60% G/C • Balanced distribution of G/C and A/T bases• Tm that allows annealing at 55-65°C• No internal secondary structures (hair-pins)
Primer pairs should have• Similar melting temperatures, Tm , within 2-3 °C• No significant complementarity (> 2-3 bp)
– particularly not in the 3’-ends
´3´3
Cycling...
Sense
Antisense
Sense
Antisense
Sense
Sense
cAntisense
Antisense
The primer dimer (PD) problem
• Primers that interact are amplified by PCR.
• PD formation competes with the designed PCR and can compromise the reaction efficiency.
Solution to the PD problem
• Reduce the formation of PDs by– Good primer design (avoid 3’ complementarity)– Minimal annealing time– Good laboratory practice– HotStart– TouchDown
• Reduce the signal from PDs by
– Measuring fluorescence above the Tm of the PDs
– Use sequence-specific probe
Considerations
• Avoid targets with secondary structure• Avoid pseudogenes• Avoid genomic contamination by designing
primers to span intron-exon-junctions
exons
introns
PCR primers
Links for designing primers
• http://www.tataa.com/• http://www.ncbi.nlm.nih.gov/BLAST/• www.premierbiosoft.com/netprimer/netprl
aunch/netprlaunch.html• www.ensembl.org • http://www-genome.wi.mit.edu/cgi-bin/pri
mer/primer3_www.cgi• http://www.bioinfo.rpi.edu/applications/mf
old/dna/form1.cgi• Primer Design- Beacon Designer/AlelleID• Primer express 3 (AB)Primer express 3 (AB)
Primer Express
• Located on Software 1 • Easy to use• Not fool-proof, but none of them
are…..
Primer design-work flow
Find sequenceFind sequence Design PrimersDesign Primers
Primer3 or similarPrimer3 or similarsoftwaresoftware
Check PrimersCheck Primers
for desired for desired parametersparameters- Tm- Tm- amplicon size- amplicon size- secondary secondary structurestructure- complementarity- complementarity- specificity- specificity
Netprimer, BLAST Netprimer, BLAST and similar and similar softwaresoftware
Satis-Satis-factory?factory?
NoNo
Run PCRRun PCRYesYes
……and gel and gel electrophoresis to electrophoresis to check specificity check specificity and functionalityand functionality
NCBI or EnsemblNCBI or Ensembl
TaqMan Probe Design
• Amplicon size 70-150 bp• Tm of probe 68-70 °C• G/C content 30-70%• No G at the 5´end• Avoid runs of identical nucleotides• Avoid secondary structure• Avoid complementarity with primers• HPLC purification
Popular dyes and quenchers
• FAM• JOE• HEX• TET• VIC• ROX• Cy
• DABCYL• TAMRA• Black Hole Quenchers
RT-PCR
• Housekeeping genes– What are they– How do you choose
• Standard curve• Primer Dimer• Melt curve• Optimization• Test samples
Reference
Workflow – preliminary data analysis
Baseline settingsBaseline - is the initial cycles in PCR where there
is little change in fluorecence signal, usually cycle ~3-15
• Set the baseline• Fixed number of cycles • Adaptive baseline
• Control baselines in the linear scale (y-axis)
Raw data
0
5
10
15
20
25
30
35
Cycles
cDNA 1:1cDNA 1:10cDNA 1:100
Baseline adjustment
0
5
10
15
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30
35
Cycles
cDNA 1:1cDNA 1:10cDNA 1:100
The different phasesExponential growth phase Plateau phase
Part of exponentialgrowth phase wheresignal > background(noise)
Samples must be compared in the exponential phase
0.01
0.1
1
10
100
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45
cDNA 1:1
cDNA 1:10
cDNA 1:100
Threshold
Setting threshold• Purpose: Find a level of fluorescence where samples can be compared• The theoretical cycle where a sample intersect the threshold is called Ct
0
5
10
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35
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43
cDNA 1:1
cDNA 1:10
cDNA 1:100
Ct
Linear
scale
Logari
thm
ic s
cale
Threshold level
Ct valuesLogLog
Setting threshold
Ct (threshold cycle): Threshold cycle reflects the cycle number at which the fluorescence generated within a reaction crosses the threshold. It is inversely correlated to the logarithm of the initial copy number
Setting threshold• Several methods available for threshold setting
– Standard deviation of the noisefor the first few cycles
– Second derivative maximum (SDM)– Best fit of standard curve (highest r2)– Manual setting
A two-fold difference in copy number should have one Ct difference no matter where the threshold is set within the exponential phase
Dilution series and standard curves
• Used to control the quality of your assays• Absolute quantification
– Standards = Diluted templates of known concentration
– Standard curve = Ct of each standard sample is plotted against the known concentration
– Used to determine concentrations of unknown samples
– Absolute quantification is dependent on the quality of the standard curve
Standard curve
0.1
1
10
100
2.00E+092.00E+082.00E+072.00E+062.00E+052.00E+042.00E+03
Comment: Always cover the whole range of sample concentrations.
0
5
10
15
20
25
30
1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08 1.E+09
Concentration (log scale)
Interpretation of the standard curve
• Linear regressionY = ax + b
a = slope that givesefficiency of PCRfrom 10–1/a = 1 + efficiency
b = # of cycles for detecting one molecule
0
5
10
15
20
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30
1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08 1.E+09
Concentration (log scale)
0
5
10
15
20
25
30
1.E+03 1.E+04 1.E+05 1.E+06 1.E+07 1.E+08 1.E+09
Concentration (log scale)
Relative quantification• Often there is no good standard available• Compare amount with reference
• Reference genes• Genomic DNA• Spike• Ribosomal RNA
• Example– Expression of target gene is 10% of the expr. of
housekeeping gene.– Same gene in other tissue, expression is 100%.
Comparing treatments
MIQUE Nomenclature
• MIQUE - Minimum Information for Publication of Quantitative Real-Time PCR experiments
Suggested nomenclature
• Reference genes not housekeeping genes• Quantification not quantitation• Hydrolysis probes not TaqMan probes• Quantification cycle Cq replaces Ct, Cp, TOP
Melting curve analysis• Melting curves are obtained by measuring
the fluorescence while increasing temperature
• Use a dye binding to double stranded DNA
70 95 Temp80 90
Melting curve analysis
• Confirms formation of the expected product (each dsDNA has its characteristic melting temp Tm)
• Distinguishes between specific PCR products and non-specific products (e.g. primer-dimers)
• High resolution melt – mutation and methylation analysis
Melting curve
0
5
10
15
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40
45
65 70 75 80 85 90 95Temperature
cyp3a high 1:1000cyp3a NTC
0
1
2
3
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7
8
9
65 70 75 80 85 90 95Temperature
cyp3a high 1:1000cyp3a NTC
Melting temperature Tm is characteristic of the %GC, length and sequence. The product can be identified from the Tm.
Tm = 90 °C
Tm = 81.5 °C
derivat
ive1st
4-steps PCR 4-steps PCR can be used to eliminate primer-dimer
signals
40 cycles
Example – 4 steps PCR
100% efficiency 75% efficiency
100% efficiency 90% efficiency
80% efficiency 50% efficiency
100%
90% 80% 75% 50%
GMNv1
3.305
3.823
4.456
4.826 7.47
VN1 3.186
3.451
3.775
3.924 4.991
Ratio 1.04 1.11 1.18 1.23 1.5
Reference primer efficiency
RT-PCR –testing samples
• ALWAYS perform melt curve• ALWAYS run negative controls
– No RT– No template
• Always Always Always run standard curve
• Triplicate of each sample!!
Requirements for RT-PCR Experiment
• Always perform standard curve• All samples in triplicate• NTC control• No RT control• Prepare mix without cDNA; add this to each tube separately• Divide plate by gene and not sample• Do not need reference gene on every plate• Melt Curve• Check RNA- otherwise don’t bother with experiment• Do not rely on only 1 reference gene- check more than one
per project• Every project is different!• Don’t be afraid to ask me questions, especially BEFORE
starting the project.
Digital PCR
From Relative quantity to absolute quantity
Commercially available machines
Fluidigm
QuantaSoft (Life Technologies)
Rain Dance
Bio Rad QX100