Software Demo

1
Stratagene Mx3000PQ-PCR Fundamentals,
Applications and Assay Design
Scott D. Leppanen Field Applications
Scientist Scott.Leppanen_at_Stratagene.com Tech.
Services 800-894-1304
2
Traditional Nucleic Acid Quantitative Methods

• Northern Blotting
• RNA size (splicing RNA integrity)
• in-situ Hybridization
• transcript localization in tissue
• RNAse Protection
• Microarrays
• large number of genes
• RT-PCR, Isothermal Amplification
• sensitive, cost effective

3
Quantitative Real Time PCR

• Definition Assay that monitors the accumulation
  of a DNA product from a PCR reaction.
• Quantitate the initial number of copies of a
  particular DNA in a sample.
• Benefits from improved sensitivity,
  reproducibility, dynamic range, throughput, cost.

4
Polymerase Chain Reaction
DNA
Gene of interest (Amplicon)
5
http//allserv.rug.ac.be/avierstr/principles/pcr.
html
6
PCR Molecular Mechanism

• Exponential amplification of the original DNA
  sequence (template) to create copies of part of
  the sequence (amplicon)

7
Quantitation of PCR product

• Limited dynamic range
• Difficult to quantitate
• Low throughput
• Not immediately amenable to replicates and stats

8
Gel-based quantification
9
Real-time quantification
10
Typical Amplification Plot
Ct
Ct Fractional PCR cycle number at which the
fluorescence intensity crosses the established
threshold line.
11
Quantitative PCR
12
Fluorescence Detection
Wavelength
13
Chemistries

• SYBR Green ITM
• TaqMan
• Molecular Beacons
• Lux primers
• Hybridization probes
• ScorpionsTM
• Amplifluor probes

dsDNA Detection
Amplicon Specific
Detection
14
Double StrandedDNA Binding Dyes
DNA free dye (weak fluorophore)
Binds minor groove dsDNA (fluorescence ?1,000x)
15
TaqMan Probes
16
Fluor-Quencher Pairs
17
Molecular Beacons
18
Lux Primers
19
LightCycler? Hybridization Probes
FRET emission
20
Scorpions
From Thelwell et al. (2000) NAR 283752
21
Amplifluor Probes
http//www.intergenco.com/pcr.html
22
Challenges of Designing a QPCR Machine
23
Considerations

• Spectral separation between excitation and
  emission wavelengths.
• Discrimination of different fluors when
  performing multiplexed assays.
• Optimize both sensitivity and dynamic range.
• Precise thermal profiles and well-to-well
  homogeneity.

24
Stratagene Mx3000PReal-Time Q-PCR Instrument
25
Stratagene Mx3000P
26
Mx3000P Thermal System

• Temperature uniformity /- 0.25 across entire
  block.
• Peltier exchange thermal block
• Heated lid with top down reading.

27
Optical System Design
Excitation filter wheel
Emission filter wheel
Halogen white light bulb
Coaxial fiberoptic cable
PMT
1 blank filter position for excitation
calibration
Read head scans over each well, filter wheels
rotate between scans
96 well plate
28
Optical System Design

• Read head takes approximately 3.5 seconds to scan
  plate
• Separate scan required for each dye selected
• Filter wheels rotate between scans

29
Excitation System

• Tungsten lamp Medium intensity, wide range.

30
Excitation Systems

• Lasers High intensity, narrow range.
• Tungsten lamps Medium intensity, wide range.
• Light emitting diodes Low intensity, defined
  wavelengths.

31
Fluorescent Wavelength Range
32
Quartz-tungsten halogen lamp excitation range
(350-830nm)
Laser excitation range
Excitation Curves
(488-514nm)
33
Detection Systems
Room temp CCD
Cooled CCD
Photodiodes
PMT
Sensitivity
34
Principles of PMTs and CCDs
e-

• Photon releases electron, positively charged
  hole remains.
• after a particular time (O10 ms), the holes on
  every pixle are counted and new electrons reset
  the chip. gt NO AMPLIFICATION

• cascade leads to exponential amplification of
  signal.
• Gain of PMT adjusts strength of amplification.
• Readout in the order of 1 ms.

35
What is the advantage of using PMTs?
16 Bit-window A/D of PMT or CCD-camera
Dye A
Dye B
Dye C
Dye D
36
Wavelength Separation
Mathematical algorithm
Filter sets
37
Fluor Excitation
- stronger fluorescence - Poor emission
discrimination
38
Fluor Emission Detection
Filter
39
Wavelength Selection
Ex.
Em.
Filter range
To optimize for sensitivity and specificity, the
excitation and emission filters of the Mx3000PTM
are slightly offset, minimizing background and
cross-talk while enhancing dye discrimination.
40
Wavelength Separation
41
Break

• Assay development
• Algorithm
• Software
• Application Specific Assays

42
Q-PCR Assay Set Up
43
Brilliant QPCR Reagents
Chose reagents for assay development and stick
with them
44
Brilliant Q-PCR Reagents
Brilliant SYBR Green Core Reagents Brilliant SYBR
Green Master Mix 1-step RT SYBR Green Master Mix
Universal Reference RNA(human, mouse, rat)
45
Assay Design
Set your primer and probe criteria and develop
your assay around a common thermal
profile http//labtools.stratagene.com
46
Amplicon Selection

• Approximate length, generally less than 400 bp
• 60-120 bp for TaqMan
• 200-400bp for SYBR
• If using a cDNA template, span exon-exon
  junctions to avoid genomic DNA contamination
  amplification
• Try to avoid repetitive or highly structured
  regions
• 5 CpG islands and control regions tend to form
  non-linear structures

47
Primer Selection

• Ideal length is 15-30 bp , Tm (Melting
  Temperature) typically 55-60C
• Taqman primers should usually have a Tm of at
  least 60C.
• Forward and Reverse primer pairs should have a
  theoretical Tm lt2 C of each other
• 40-60 GC nucleotide content is ideal prevent G/C
  regions self-hybridizing.
• If requested by the design software, use 100 mM
  monovalent cation and 5 mM Mg.
• Always BLAST your primers.

48
TaqMan Probe Selection

• Ideal 20-30 bp in length
• Tm 10C higher than primers
• 35-65 G/C more Cs than Gs..
• Avoid runs of gt3 of the same nucleotide,
  especially Gs.
• 5 base ? G.
• Primer Probe distance should be 3-12 bp
• Test that primers and probe are not complementary
  to each other
• delta G free energy at 25C should be greater than
  -2 for singleplex rxn

49
Primer Optimization
TaqMan may require more of the same primers for a
SYBR Green assay.
50

Primer dilutions
Scott pr opt SYBR Green, 02-18-2004.mxp
51

Primer dilutions
Scott pr opt SYBR Green, 02-18-2004.mxp
52
Primer Optimization
53
Primer Optimization
Single product
54
Atypical Primer Behavior
55
Template Dilutions for Assay Validation
Efficiency 100
Efficiency gt 100
Ct
Log (Quantity)
56
Calculation of Efficiency
57
Quantitative Algorithm

• Correct for noise in signal measurement
• Normalize all raw sample data to zero on the
  y-axis
• Establish a threshold to separate noise from true
  fluorescent signal
• Threshold needs to be optimal for all samples
  being compared within a run

58
Quantitative Algorithm
59
Moving Average Algorithm
In order to minimize noise in the data,
fluorescence values can be smoothed by replacing
each data point with the average of the points
around it. (3, 5, 7, 9 pt user selectable)
60
Optional Data Smoothing
61
Adaptive Baseline Algorithm
62
Baseline Subtraction
35000
R
30000
R 1
25000
20000
R
15000
10000
5000
0
0
10
20
30
40
-5000
Cycle
63
Adaptive Baseline

• Based on the shape of the raw data (R), optimal
  baseline cycles are defined for each well and
  fluor.

64
Amplification Based Threshold
65
Threshold Value

• Separates the data from the noise.
• Valid for Ct calculations if placed during
  exponential amplification.
• 3 Options
• Default (Minimizes variability between
  replicates)
• 10 times the noise during early cycles.
• Manual (click and drag)
• On exponential phase.
• Lines are parallel.
• Minimize variability.

66
Reference Dye Normalization
Correct for well to well artifacts in fluorescence
67
Reference Dye Normalization
dRn, Rn
68
Display Formats
69
Software OrganizationFollows the logic of an
experiment
70
Question about a particular screen or set up?

• Click F1 while on that screen

71
Quick Setup Wells can be defined before or after
the run.
72
Point-and-Click PCR Profile Setup
73
Run Status Box Monitors the Progress of the Run
74
Run Status Dialog Box
75
Real-time Amplification Data
76
Mx3000P Applications

• Any time a specific sequence is detected or
  quantified
• Qualitative Detection.Pathogens, GMO,
  environmental testing, quality control.
• Relative and comparative quantification
• Gene expression, microarray validation
• Absolute quantification.(Viral load , genomic,
  mitochondrial DNAs, quality control).
• Sequence Detection
• Allele discrimination, genotyping, SNPs, zygosity
  testing, methylation, etc.
• Fluorescence Detection
• Isothermal signal amplification, plate reader
  functionality.

77
Qualitative Sequence Detection
HSV unknowns (FAM), prepared with internal
positive controls (TET)
78

Qualitative Sequence Detection
79
Standard Curve
80
Relative Quantitation
81
Allele Discrimination Plot
82
Application-Specific Experiment Types
83
Troubleshooting the Baseline
84
Amplification plots Semi-log plot
85
Absolute Quantification, Ct vs DNA
86
Comparative Quantification

• Relative quantification
• Experimental samples are compared to a dilution
  series of a control template source using the
  same target(s).
• Assumes is that the template yield is constant
  (cDNA synthesis or RNA obtained per cell or mg of
  tissue).

• Templates for relative quantification
• Viral RNA from control samples or tissue (must
  contain the target at detectable levels).
• T7 transcripts from cloned amplicon.
• Purified PCR product.
• Synthetic oligonucleotide.
• Universal RNA.

87
Comparative Quantification
Given two samples What is the difference in
target concentration?
88
Comparative Quantification

• Purpose
• Determine relative expression levels between two
  samples.
• Samples
• Unknowns or experimentals.
• Calibrator or Reference sample Any sample with a
  constant GOINormalizer ratio
• Control tissue or cells.
• Standard cell line RNA.
• Universal Reference RNA or cDNA.
• Targets
• Gene(s) of interest.
• Normalizer Constant levels per cell across all
  samples
• Housekeeping or other constant expression gene.
• Exogenous RNAs spiked at RNA purification.

89
Example IL-1 expression in cells following
stimulation. GAPDH used as normalizer. Efficiency
at 100 for both targets ? (1 Eff ) 2 Control
samples yield higher RNA
90
Comparative Quantification Setup
Separate wells
91
Relative Quantitation
92
Absolute Quantification

• Absolute quantification
• Relative to a standard set of known concentration
  (purified virus, cloned targets, etc.).
• Assumes equal efficiency of amplification between
  unknowns and standards.
• Must run a standard in every plate.
• Results readily comparable to other techniques or
  sources.

93
PCR Rxn Efficiency (E) 10-1/slope - 1
Good
94
Standard Curve
95
Normalizing Comparative Quantification Results
Quantity
Gene of Interest (FAM labeled)
Normalizer (HEX labeled)
96
Qualitative Sequence Detection

• Pathogen (Bacterial/Viral) Load
• Single endpoint read
• Plate Read format
• Yes/no determination
• Amplification plot/threshold based
• Comparative Quantitation (Calibrator)
• Better discrimination of signal due to sequence
  of interest, compared to noise
• Can normalize to host sequence

97

Qualitative Sequence Detection
98
SYBR Green I Detection
99

Dissociation Curve Analysis
100
Replicate Averaging

• Treat individually ignores replicate numbers
• Treat collectively averages the raw data
  according to the assigned replicate number.
• Data normalization, baseline, etc. are
  recalculated.
• Considerations
• Outliers may introduce a bias in the analysis
  results.
• Loss of information about statistical variation.

101
Replicate AveragingTreated individually
102
Replicate AveragingTreated collectively
103
Melt Curve de novo SNP Detection
104

Melt Curve de novo SNP Detection
105
Molecular Beacon SNP Detection
106
Allele Discrimination Plot
107
Qualitative detection
108
Export Chart Data
109
Export Results in Multiple Formats
Text Report
110
Access to raw data
111
Performance Data

• QC Validation Run Specifications
• Uniformity run CV lt 0.8
• Two example uniformity runs (HEX-labeled
  molecular beacon and SYBR Green I)
• Two-fold serial dilutions
• 4-way multiplex data.

112

Uniformity Amplification Plots
113

Uniformity Amplification Plots
96 wells Mean Ct 18.1 St.Dev. 0.05 C.V .
0.3 Ct Range 0.26 cycles
114

Dilution Series Amplification Plot
2 fold dilutions, triplicate, 2-20,000
copies
115

Standard Curve
Std. Curve R2 0.996 Slope -3.183 Amplification
Efficiency106.1
116

4-Color Multiplex
117

4-Color Multiplex-Standard Curves Together
E Cy595.3 E Rox98.9 E Hex101.3 E Fam91.7
118

4-Color Multiplex-Standard Curves Alone
E Cy595.1 E Rox97.7 E Hex94.7 E Fam98.8
119
Practical Considerations

• Good quality template is necessary
• Diluted in water, high integrity RNA if doing RT
• Take care to design/maintain good reagents
• Primer design software, BLAST, test, optimize
• Avoid multiple freeze thaws, store DNA in TE.
• Avoid high copy number areas-cloning
• Use a reference dye, powder free gloves
• Run replicates, track your data, statistics
• Efficiencies, R2 fit, Std Dev

120
Data Quality
121
Data Quality
122
For Further Advice

• Search PubMed with terms RT-QPCR
• Contact Stratagene Technical Services
• 800-894-1304, Pacific Standard Time
• QPCRSystemsSupport_at_Stratagene.com
• Regular Webinars covering PCR topics
• Primer and Probe Design
• Multiplexing
• Advanced Data Analysis, etc
• Check www.Stratagene.com/Events