Dr Abida Raza

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Real Time PCR

• Dr Abida Raza
• Senior Scientist
• Molecular Diagnostics Research Laboratory
• Nuclear Medicines Oncology Radiotherapy
  Institute

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Outline

• 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

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Cockeril FR III. Arch Pathol Lab Med.
20031271112 (www)
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Limitations 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

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Why 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

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Development 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.

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Real-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

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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)
• 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

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Real-time PCR disadvantages

• Not ideal for multiplexing
• Setting up requires high technical skill and
  support
• High equipment cost

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Real-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

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Real 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
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Real-Time and End Point
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Linear Vs Log View
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Exponential growth phase linear part in
logarithmic graphic
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Real 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)

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Hydrolysis 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.

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dNTPs
Primers
Add Master Mix and Sample
Thermal Stable DNA Polymerase
Reaction Tube
Denaturation
l
Annealing
Taqman Technology
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Taqman Technology
5
3
Extension Step
1. Strand Displacement
2. Cleavage
3. Polymerization Complete
l
4. Detection
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Hybridization 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.

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SYBR 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.

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SYBR 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

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When 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

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When 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

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TaqMan probes
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Molecular Beacons
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FRET probes
LUX fluorogenic primers
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9
MGB Eclipse probes
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Other
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Scorpion probes
0
10
20
30
40
50
60
70
80
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Design 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

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One-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

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Selection of method
Kits
Components

• Greater flexibility
• Less expensive
• Have own system
• Too little reagent volume in kits

• Convenience
• Guaranteed optimized system
• Cost effective

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Selection of Instrument
Performance Parameters
Product Specifications

• Sensitivity
• Linear dynamic range
• Time-to-results
• Throughput
• Excitation Source

• Software
• Block/sample capacity
• Size
• User interface

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• 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.

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What 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

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Services offered by NORI

• Hepatitis C and Hepatitis B
• Qualitative Tests, negative/positive
• Quantitative TestingViral load
• HCV genotyping
• HBV genotyping

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HCV 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?

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HCV 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

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Molecular 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.

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Assessment 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.

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Clinical 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

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Real 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
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Quantitation 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
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• 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        
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• HBV

No. 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            
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• 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        
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Quality 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
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• 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      
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• 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  
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Accuracy 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
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Use 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
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• 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

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• 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.

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Efficiency

• 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

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Using 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
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DRn
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
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Applications 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.

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Thank you