Title: Dr Abida Raza
1Real Time PCR
- Dr Abida Raza
- Senior Scientist
- Molecular Diagnostics Research Laboratory
- Nuclear Medicines Oncology Radiotherapy
Institute
2Outline
- Conventional PCR
- Real Time PCR
- Chemistries of Real Time PCR
- How to develop the assays
- Use of Real Time PCR at NORI
- Quality Control issues
- Application of Real Time PCR
3Cockeril FR III. Arch Pathol Lab Med.
20031271112 (www)
4Limitations of Conventional PCR
- Assumptions on reaction consistency and
uniformity - Narrow dynamic range
- Long optimisation and set up times
- Long run and analysis times
- High levels of inherent inaccuracy and variation
- PostPCR detection procedure
- Low detection limit
5Why Real Time PCR amplification
- Question from Yes or No to How and/or How many?
- Qualitative PCR to Quantitative PCR
-
- Regulation of gene expression
- Disease diagnosis
- Therapeutic monitoring
- Contamination issues
- Automation issues
- Limitation of conventional PCR
6Development of Real Time Analysis
- First reported in 1992 by Higuchi et al.
- Used ethidium bromide to intercalate into double
stranded (ds) DNA and a thermal cycler modified
with a cooled charged coupled device (CCD camera)
attached. - PCR cycle dsDNA dye fluorescence
- Later changed to SYBR Green I as this has a much
higher affinity for dsDNA rather than ssDNA
compared to ethidium bromide.
7Real-Time PCR
- Real-time PCR monitors the fluorescence emitted
during the reaction as an indicator of amplicon
production at each PCR cycle (in real time) as
opposed to the endpoint detection
8Real-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)
- Ultra-rapid cycling (30 minutes to 2 hours)
- Wider dynamic range of up to 1010-fold
- Requirement of 1000-fold less RNA than
conventional assays - (6 picogram one diploid genome equivalent)
- Detection is capable down to a two-fold change
- Confirmation of specific amplification by
melting curve analysis - Most specific, sensitive and reproducible
9Real-time PCR disadvantages
-
- Not ideal for multiplexing
- Setting up requires high technical skill and
support - High equipment cost
10Real-time PCR Principles
- Based on the detection and quantitation of a
fluorescent reporter - The first significant increase in the amount of
PCR product (CT - threshold cycle) correlates to
the initial amount of target template
11Real Time PCR
Real-time PCR monitors the fluorescence emitted
during the reaction as an indicator of amplicon
production at each PCR cycle (in real time) as
opposed to the endpoint detection
Theoretical
Amplification is exponential, but the exponential
increase is limited
Log Target DNA
Real-Time PCR allows us to see the exponential
phase so we can calculate how much we started
with.
CT
Cycle
12Real-Time and End Point
13(No Transcript)
14Linear Vs Log View
15Exponential growth phase linear part in
logarithmic graphic
16Real Time PCR Chemistries
- Three general methods for the quantitative
assays - 1. Hydrolysis probes (TaqMan, Beacons)
- 2. Hybridization probes, (Light Cycler)
- 3. DNA-binding agents (SYBR Green)
17Hydrolysis probe technique
- The hydrolysis probe is conjugated with a
quencher fluorochrome, which absorbs the
fluorescence of the reporter fluorochrome as long
as the probe is intact. - Upon amplification of the target sequence, the
hydrolysis probe is hydrolyzed by the Taq
polymerase resultings in the separation of the
reporter and quencher fluorochrome - Consequently the fluorescence of the reporter
fluorochrome becomes detectable. During each
consecutive PCR cycle this fluorescence will
further increase because of the progressive and
exponential accumulation of free reporter
fluorochromes.
18dNTPs
Primers
Add Master Mix and Sample
Thermal Stable DNA Polymerase
Reaction Tube
Denaturation
l
Annealing
Taqman Technology
19Taqman Technology
5
3
Extension Step
1. Strand Displacement
2. Cleavage
3. Polymerization Complete
l
4. Detection
20Hybridization probes technique
- One probe is labelled with a donor fluorochrome
at 3 end and a second adjacent- probe is
labelled with an acceptor fluorochrome. When
two fluorochromes are in close vicinity (15 nt
apart), the emitted light of the donor
fluorochrome will excite the acceptor
fluorochrome (FRET) resulting in the emission of
fluorescence, which can be detected during the
annealing phase and first part of the extension
phase of the PCR reaction. After each subsequent
PCR cycle more hybridization probes can anneal,
resulting in higher fluorescence signals.
21SYBR Green technique
- SYBR Green fluorescence is enormously increased
upon binding to double-stranded DNA. During the
extension phase, more and more SYBR Green I will
bind to the PCR product, resulting in an
increased fluorescence. Consequently, during each
subsequent PCR cycle more fluorescence signal
will be detected.
22SYBR Green
- (double-stranded DNA binding dye)
- Emits a strong fluorescent signal upon binding to
double-stranded DNA - Nonspecific binding is a disadvantage
- Requires extensive optimization
- Requires melting point curve determination
- Longer amplicons create a stronger signal
- May be multiplexed when coupled with melting
curve analysis
23When to Choose SYBR Green
- Assays that do not require specificity of probe
based assays. Detection of 1000s of molecules - General screening of transcripts prior to moving
to probe based assays - When the PCR system is fully optimized -no primer
dimers or non-specific amplicons, e.g. from
genomic DNA
24When Not to Choose SYBR Green
- Allelic discrimination assays (not an absolute
one) - Multiplex reactions (not an absolute one)
- Amplification of rare transcripts
- Low level pathogen detection
2578
TaqMan probes
19
Molecular Beacons
15
FRET probes
LUX fluorogenic primers
9
9
MGB Eclipse probes
3
Other
2
Scorpion probes
0
10
20
30
40
50
60
70
80
26Design the experiment What you need?
- I. Assay Development
- A. Sequence selection
- B. Primer probe selection
- C. Quencher dye and internal reference
- D. Assay validation
- II. Assay Setup
- A. One- or two-step PCR
- B. Thermocycler settings
- III. Data Analysis
- A. Baseline and threshold settings
- B. Standard curves
- C. Inter- vs intra-assay variability
- D. Sample normalization
27One-Step or Two-Step PCR
- One-step real-time RT-PCR performs reverse
transcription and PCR in a single buffer system
and in one tube - Two-step RT-PCR performs reverse transcription
and PCR in different tubes
28Selection of method
Kits
Components
- Greater flexibility
- Less expensive
- Have own system
- Too little reagent volume in kits
- Convenience
- Guaranteed optimized system
- Cost effective
29Selection of Instrument
Performance Parameters
Product Specifications
- Sensitivity
- Linear dynamic range
- Time-to-results
- Throughput
- Excitation Source
- Software
- Block/sample capacity
- Size
- User interface
30- The majority of researchers prefer using kits
rather than individual components for real-time
PCR amplification. - TaqMan probes are the most popular choice for
users of fluorescent probes/primers. - Software, sensitivity and user interface are the
most important features of real-time PCR
instrumentation.
31(No Transcript)
32What you want to do?
- Absolute Quantitation
- Standard Curve
- Standards must be accurately quantified
- Best for viral load determination like Hep C, Hep
B - Relative Quantitation
- Standard Curve
- Standards are serial dilutions of calibrator
template - Best for gene expression studies, like Her2 neu
gene expression - Comparative Quantitation
- Mathematical Determination
- Calibrator sample used as 1x standard
- Best used when particular ratios are expected or
to verify the trends
33Services offered by NORI
- Hepatitis C and Hepatitis B
- Qualitative Tests, negative/positive
- Quantitative TestingViral load
- HCV genotyping
- HBV genotyping
34HCV Infection- An Example
- Already available/in use techniques
- Biological markers
- Virological markers
HCV Molecular Diagnostics! Why ?
- Diagnostic testingYes/NoPCR
- Treatment selection/follow up of
progressionPathogen concentration?
35HCV Molecular Diagnostics
- Acute Hep C
- Detection of HCV-RNA (50 HCV RNA IU/ml or less)
without anti-HCV------strong indicative - Chronic Hep C
- Both anti-HCV HCV-RNA (50 HCV RNA IU/ml or
less) - Anti-HCV positive, but HCV RNA is undetectable
for at least two occasions 6 months apart, it is
very difficult to distinguish patients who still
harbor antibodies after spontaneously resolving
HCV infection in the past from patients with
false-positive reactivity
36Molecular Diagnostics helps in treatment decision
schedule
- Genotype 1---only 40-45 chance of responding to
therapy48 week of treatment---1.0-1.4 g
ribavirin qd - Genotyping 2 or 3---70-80 response--24 weeks of
therapy---0.8g ribavirin qd - Baseline HCV RNA quantification must be performed
in genotype 1 infected patients, it serves as
reference value to assess virologic response at
week 4,12 and 24. - Same is the case for genotype 4, 5 and 6.
37Assessment of virological response to therapy
- If level is undetectable at week 4, Patient is a
rapid virological responder, more chance of SVR - If HCV RNA is undetectable at week 12, patient is
regarded as early virological responder - If 2 log drop is observed at week 12 patient is
called partial early virological responder,
chances of SVR decreased. - If HCV RNA is detectable at week 24, patient is
called non responder, may be asked for prolong
treatment for 48 week - If HCV RNA is negative at week 24, but positive
after 24 week of completion of therapy, patient
is a relapse case.
38Clinical Oncology
- Her2/neu detection
- Amplification/strong expression can be seen in 20
to 30 invasive breast carcinomas - Marker of adverse clinical outcome
- Predictive marker for reduced response to therapy
hormonal treatment - Positive Her-2 status predicts response to
Herceptin
39Real Time PCR based Diagnostics
- 1. Blood Sample Collection
- (Plasma stored at -20C)
- 2. RNA/DNA Extraction using King Fisher/Manual
4.Setting up of Rotor Gene for detection
5. Analysis of run using Corbett Research 6000TM
Software
3. Master Mix Prep
40Quantitation Report Quantitation Report
Experiment Information Experiment Information
Run Name Run 2008-12-02 (1)
Run Start 12/2/2008 834
Run Finish 12/2/2008 1129
Operator Dr Abida
Run On Software Version Rotor-Gene 6.0.25
Run Signature The Run Signature is valid.
Gain FAM/Sybr 5
Gain JOE 5
Gain ROX 5
Gain Cy5 5
Col Name Type Ct Given Conc (IU/ml) Calc Conc (IU/ml)
Std-1 Standard 12.29 6,000,000,000 6,000,000,000
NTC(629) NTC
676 Unknown 21.64 10,285,706
677 Unknown 28.8 78,410
679 Unknown 23.61 2,695,607
680 Unknown 29.69 42,704
681 Unknown
682 Unknown 23.37 3,160,855
683 Unknown 31.81 10,057
684 Unknown 23.74 2,463,616
685 Unknown 21.35 12,533,848
686 Unknown 36.08 551
687 Unknown 23.7 2,536,686
689 Unknown 25.3 849,671
690 Unknown 28.01 134,133
692 Unknown 24.86 1,144,266
693 Unknown 24.25 1,733,457
694 Unknown 26.04 514,578
695 Unknown 24.76 1,225,342
697 Unknown
698 Unknown
699 Unknown 23.9 2,212,108
701 Unknown 26.45 388,918
702 Unknown 30.95 18,170
703 Unknown 22.6 5,365,739
Positive C Unknown 27.33 212,960
(Cy5,FAM/Sybr,JOE,ROX)
Viral load Expressed as IU/ml Low less than
8x105 IU/ml High more than 8x105 IU/ml
41- HCV
- Viral Load Calculations of Replica Ct with
Standard Deviation -
No. Name Ct Given Conc (IU/ml) Calc Conc (IU/ml) Rep. Ct Rep. Ct Std. Dev. Rep. Ct (95 CI) Rep. Calc. Conc.
1 Std-1 12.51 6,000,000,000 6,462,821,584 12.61 0.13 12.41 , 12.81 6,000,000,000
2 Std-1 12.57 6,000,000,000 6,170,814,416
3 Std-1 12.8 6,000,000,000 5,297,215,788
4 Std-1 12.58 6,000,000,000 6,134,690,889
5 NTC
Sr Name Ct Given Conc (IU/ml Calc Conc (IU/ml) Var
1 STD1 11.24 6,000,000,000 6,000,974,435 0.00
2 STD3 18.21 60,000,000 59,993,398 0.00
3 STD5 25.18 600,000 599,771 0.00
4 STD7 29.72 30,000 30,010 0.00
5 NTC
42No. Name Type Ct Calc Conc (IU/ml) Var Rep. Ct Rep. Ct Std. Dev. Rep. Calc. Conc.
1 STD 2 Unknown 22.55 2,070,488 3.90 22.60 0.15 2,026,196
2 STD 4 Unknown 27.85 52,021 4.00 27.87 0.11 50,335
3 STD 6 Unknown 31.28 5,933 2.30 31.21 0.11 5,940
4 STD 2 Unknown 22.66 1,985,416 4.70
5 STD 4 Unknown 27.90 49,911 5.20
6 STD 6 Unknown 31.04 5,950 2.00
7 NTC Neg cont
43- Her2/neu
- Quantitative gene expression data is normalized
to the expression levels of control or so-called
"housekeeping"gene GAPDH Glyceraldehyde-3-phospha
te dehydrogenase. Result is taken as ratio of
Her2/neu to GAPDH
No Name Type Ct Given Conc (IU/ml) Calc Conc (IU/ml) Var
1 STD1 Standard 19.33 100,000 99,966 0.00
2 STD2 Standard 23 10,000 10,004 0.00
3 STD4 Standard 27.77 500 500 0.00
4 STD7 Standard 32.53 25 25 0.00
7 Neg. cont NTC
44Quality Control Issues
- Instrument sensitivity single copy
- Technology selection Taqman/Dual labeled
probe/FRET - Method/kit sensitivity 172 IU/ml
- 50 IU/ml (95 confidence interval)
- 4 controls in each run---
- Reproducibility/repeatability/precision
- Random use of Negative and positive samples in
random runs, intra run STD variation
Sr . Controls Type Monitoring output
1 Internal Control Gene other than gene of interest how good is extraction
2 Known Standards 8 known values-- 6x109, 6x108, 6x107, 6x106, 6x105, 6x104, 3x104, 6x103 IU/ml how good is pipetting, how accurate/close to standard curve ,ultimately patient viral load figures (accuracy)
3 Positive Control WHO standard, genotype 1 --80,000 IU/ml helps in having check on accuracy/precision of run and ultimately in patient viral load values.
4 Negative Control Healthy Sample contamination free area
45- Reproducibility/repeatability/precision
- Random use of Negative and positive samples in
random runs, intra run STD variation
No. Name Ct Given Conc (IU/ml) Calc Conc (IU/ml) Rep. Ct Rep. Ct Std. Dev. Rep. Calc. Conc.
19 5376 25.91 Unknown 11,160 25.96 0.08 10,761
20 95 16.99 Unknown 4,895,610 16.97 0.02 4,949,705
21 5376 26.02 Unknown 10,376
22 95 16.96 Unknown 5,004,397
46- Accuracy
- No. of true positives No. of true
negatives/No. of true positives false positives
false negatives true negatives - must be 100 for Dual Labeled Probe.
Sr Name Ct Given Conc (IU/ml Calc Conc (IU/ml) Var
1 STD1 11.24 6,000,000,000 6.008.344,001 0.30
2 STD3 18.21 60,000,000 60,401,240 0.70
3 STD5 25.18 600,000 598,004 0.30
4 NTC
5 459 22.62 Unknown 10,339,876
6 459 Dup 22.59 Unknown 10,534,415
47Accuracy and Precision calculations
Accuracy (inter run) inter standards 99.98
0.015 Precision (intra runs)
Name age precision
STD 1 100.00 0.40
STD 2 99.29 0.29
STD 3 99.26 0.03
STD 4 99.24 1.01
STD 5 99.25 0.89
STD 6 98.94 1.12
STD 7 97.511.33
STD 8 98.59 1.03
48Use of Internal control
Quantitation data for Cycling A.FAM/Sybr HCV 629
as negative control
STD
Patient
No. Colour Name Ct Given Conc (IU/ml) Calc Conc (IU/ml)
1 Std-1 12.29 6,000,000,000 6,000,000,000
2 NTC
3 Unknown 21.64 10,285,706
Neg. C
Quantitation data for Cycling A.JOE HCV-676
HCV-629
Neg. C
No. Colour Name Ct Given Conc (IU/ml) Calc Conc (IU/ml)
1 Std-1 33.0 6,000,000,000 6,000,000,000
2 NTC ) 34.0 3,063,326,785
3 Unknwon 33.8 3,401,134,851
Patient
STD
49- Parameters to be kept in mind during ANALYSIS
- Slope correction
- Dynamic tube settings
- Intra standard age variance calculated
automatically within run (Accuracy) - Intra run age variance (Precision) calculated
manually - Threshold settings, calculated manually keeping
in view the actual Well Fitted Data. - Threshold cycles Statistically significant
fluorescence as compared to back ground - Elimination of early cycles, Difference Of
Handling
50- R2 value of standard curve, must be Between 1 to
0.9 - r value of the run, which is a measure of how
well the Actual Data Fit To The Standard Curve. - r (explained variation/total variation)
- Slope value of the run must be within a certain
range/value (-3.76 to -3.04) - Efficiency of the reaction
- Efficiency (h) 10(-1/slope) 1
- Most important calculations or a measurement can
be accurate but not precise, precise but not
accurate, neither, or both. A measurement system
or computational method is called valid if it is
Both Accurate And Precise. -
51Efficiency
- The slope of the log-linear phase is a reflection
of the amplification efficiency - The efficiency of the PCR should be 90-110
- (ideal slope 3.32)
- A number of variables can affect the efficiency
of the PCR. These factors can include length of
the amplicon, secondary structure and primer
design
52Using the PCR Equation
- Xn X0(1 E)n
- Xn PCR product after cycle n
- X0 initial copy number
- E amplification efficiency
- n cycle number
- If the CT values for each of the dilutions are
plotted against concentrations, the result should
be a linear graph with a high correlation
coefficient (gt 0.99)
Xn
X0
cycle number
53DRn
DRn is the difference between Rn and Rn-. It
is an indicator of the magnitude of the signal
generated by the PCR Rn is the Rn value of a
reaction containing all components (the sample of
interest) Rn- is the Rn value detected in NTC
(baseline value) DRn is plotted against cycle
numbers to produce the amplification curves and
to estimate the CT values
54Applications Of Real-Time PCR
- Quantitative Real time PCR is currently used in a
vast array of applications in both academic and
diagnostic research. - Diagnostic real-time PCR
- Detect nucleic acids that are diagnostic, e.g.
infectious diseases, cancer and genetic
abnormalities. - Clinical microbiology like HCV
- Emerging disease like swine Flu
- In research settings
- Mainly used to provide quantitative measurements
of gene transcription. - May be used in determining how the genetic
expression of a particular gene changes over
time, such as in the response of tissue and cell
cultures to an administration of
a pharmacological agent, progression of cell
differentiation, or in response to changes in
environmental conditions.
55Thank you