Real-Time-PCR

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Biochemistry

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Real Time PCR:

Real Time PCR M.Prasad Naidu MSc Medical Biochemistry, Ph.D ,.

Traditional PCR (semiquantitative):

Traditional PCR (semiquantitative) annealing 60°C denaturation 95°C extension 72°C Gel electroforesis 1 2 4 8 2 n n cycles Specificity determined by 2 primers

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Amplification Plot of 96 Sample Replicates Precise C t Variable PCR Plateau (taq efficiency decreases, reagents get limiting, decreased denaturation efficiency, …) Traditional PCR (semiquantitative) Plateau phase Plateau phase

Principles of quantitative PCR:

Principles of quantitative PCR Monitors the progress of the PCR as it occurs (in “real time”) by reading fluorescence intensities after each cycle. Intensities are proportional to the number of amplicons generated Samples are characterized by the point in time during cycling, when amplification is first detected (more starting material  sooner an increase in fluorescence ) For allelic discrimination, endpoint assays are used Temperature protocol: also > 30 cycles 30” 95°C 30” 60°C 30” 72°C/60°C

Exponential growth phase = linear part in logarithmic graphic:

Exponential growth phase = linear part in logarithmic graphic 1 2 4 8 2 n n cycles

CHEMISTRY:

CHEMISTRY SYBR green Taqman probes Molecular Beacons Scorpion primers

1) SYBR green I:

1) SYBR green I intercalating dye, binds double strand DNA More sensitive than EtBr Specificity determined by 2 primers No probe required (lower costs) Also detection of aspecific products  m elting curve after PCR reaction Only singleplex No allelic discrimination possible CHEMISTRY

Dissociation Curve:

Dissociation Curve Dissociation Protocol can be added to the thermal cycling parameters Allows detection of non-specific products cycle 40 60°C 1min 95°C 15s 60°C 20s 95°C 20min 1) SYBR green I CHEMISTRY

Dissociation Curve:

Raw Data View Natural decrease in SYBR fluorescence SYBR dissociates from ds amplicon baseline Tm = temperature when 50% dissociated Dissociation Curve

Derivative Data View:

Target amplicon Derivative Data View Tm = temperature when 50% dissociated Dissociation Curve

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Example: Presence of Primer Dimers Dissociation Curve Product Primer dimers or aspecific product

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Mechanism : fluorescence resonance energy transfer (FRET) Cleavage (5’ nuclease activity of taq DNA polymerase) Increase of reporter signal proportional to amount of amplicon produced Removes probe from target strand Annealing and polymerization Strand displacement Cleavage 2) Taqman probe CHEMISTRY

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Two primers + a fluorogenic probe determine specificity No detection of aspecific products No melting curve needed (faster) Can be used for allelic discrimination Multiplex Synthesis of different probes required for different sequences 2) Taqman probe CHEMISTRY

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Reporters: FAM,TET,VIC,JOE Quenchers: TAMRA, MGB Passive reference: ROX Emission Profiles of Various Fluorophores: Fam Vic/Joe Tamra Rox Multiplex reactions possible 2) Taqman probe CHEMISTRY normalizes for non-PCR-related fluorescence fluctuations occurring well-to-well (concentration or volume differences)  Spectral compensation necessary

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Baseline Threshold Rn Ct Baseline = Basal level of fluorescence defined during the initial cycles of PCR (background fluorescence). Threshold = Fixed fluorescence level set above the baseline (statistical cutoff based upon background fluorescence). Rn = normalized Reporter signal, level of fluorescence detected during PCR. Calculated by dividing probe reporter dye signal by passive reference signal (ROX). Ct = threshold Cycle, PCR cycle at which an increase in reporter fluorescence above a baseline signal is first detected (cycle when fluorescence crosses the threshold). DEFINITIONS

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Setting baseline and threshold (exponential growth)  determining Ct (threshold cycle) of each sample Ct is the cycle number at which the fluorescence passes the threshold EXAMPLE GRAPHIC Ct Ct Ct Ct DEFINITIONS threshold baseline

Advantages of using Real-Time PCR:

Advantages of using Real-Time PCR COLLECTS DATA IN THE EXPONENTIAL GROWTH PHASE REAL TIME : permanent record of amplification INCREASED DYNAMIC RANGE of detection LESS RNA NEEDED Requirement of 1000-fold less RNA than conventional assays FAST : No-post PCR processing SENSIBLE : Detection is capable down to a 2-fold change

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Real time detection: Quantitation of gene expression Quantitation of RNA, DNA, cDNA Viral quantitation … Endpoint detection: Allelic discrimination (SNP genotyping) Plus/minus studies Pathogen detection … APPLICATIONS

PowerPoint Presentation:

Real time detection: Quantitation of gene expression Quantitation of RNA, DNA, cDNA Viral quantitation … Endpoint detection: Allelic discrimination (SNP genotyping) Plus/minus studies Pathogen detection … APPLICATIONS

Quantification:

Quantification Absolute quantification (result in copy number): virus copy number,… 1. Calculation by standard curve Relative quantification (result is given as relative to the reference sample): gene expression,… 2. Calculation by standard curve 3. Use of comparative Ct method

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Real time detection: Quantitation of gene expression Quantitation of RNA, DNA, cDNA Viral quantitation … Endpoint detection: Allelic discrimination (SNP genotyping) Plus/minus studies Pathogen detection … APPLICATIONS

RNA reverse transcription:

RNA reverse transcription

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Absolute Quantitation: Standard Curve

Standard curve:

Standard curve Quantify sample by spectrofotometry, make dilution curve

Standard curve:

Ct Log Qty Ct= 29.7 Log Qty = 3.28 Standard curve

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Dynamic range

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Relative Quantitation: Standard Curve

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Cells: Basal conditions Treatment IL6 3h Treatment OSM 3h Define expression of gene of interest (SOCS3) upon treatment, relative to expression at basal conditions Relative quantitation: example

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β -actin GAPDH 18S and others ratio target gene (experimental/control) = fold change in target gene (exp/control)   fold change in reference gene (exp/control) The perfect standard does not exist; choose the best control for your system We need an endogenous control to normalize for the amount of starting material in the tube !

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Make dilution series of a sample Read SOCS3 levels of standard curve and unknown samples Read 18S levels of standard curve and unknown samples; Choose a sample (cells in basal conditions) as calibrator If level SOCS3 (sample IL6 45 min)/ level SOCS3 (sample basal conditions)= 10x Level 18S (sample IL6 45 min)/ level 18S (sample basal conditions)= 2x  Then the level of SOCS3 after IL6 treatment is 5x higher than at basal conditions Relative quantitation: example

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Relative Quantitation: ΔΔ Ct method

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Principle: Samples that differ by a factor of 2 in the original concentration would be theoretically expected to be 1 cycle apart. Samples that differ by a factor of 10 (as in our dilution series) would be ~3.3 cycles apart. CT (sample) - CT (basal) Relative Quantity = 2 Example 1: Ct(A)= 30 Ct(B)= 31 RQ = 2 1 = 2 Example 2: Ct(A)= 30 Ct(B)= 33,3 RQ = 2 3.3 = 10 ΔΔ Ct method

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 CT(sample) = CT (Target) - CT (Reference)  CT(calibrator) = CT (Target) - CT (Reference)   CT =  CT (Sample) -  CT (Calibrator) Relative Quantity = 2 - ΔΔ Ct ratio = fold change in target gene (sample) fold change in reference gene (sample) fold change in target gene (calibrator) fold change in reference gene (calibrator) BUT: 8 targ 4 ref 12 targ 2 ref sample calibrator

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tr 18S ctrl SOCS3 tr SOCS3 ctrl SOCS3 tr SOCS3 ctrl 18S ctrl 18S tr 18S Example 1 :  CT(sample) = CT (Target) - CT (Reference) D Ct (ctrl SOCS3)= 27-20 = 7 D Ct ( tr SOCS3)= 24-20 = 4  CT =  CT (Sample) -  CT (Calibrator) DD Ct = 4 – 7 = -3 RQ = 2 3 = 8 Note: Also Ct( tr SOCS3) – Ct(ctrl SOCS3) = 27-24= 3 because starting conc was equal (equal 18S)  SOCS3 expression in treated sample is 8 times higher than in control sample. ΔΔ Ct method: Example

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tr 18S ctrl SOCS3 tr SOCS3 ctrl SOCS3 tr SOCS3 ctrl 18S ctrl 18S tr 18S Example 2 :  CT(sample) = CT (Target) - CT (Reference) D Ct(ctrl SOCS3)= 28-16 = 12 D Ct(tr SOCS3)= 25-13 = 12  CT =  CT (Sample) -  CT (Calibrator) DD Ct = 12 – 12 = 0 RQ = 2 0 = 1  no difference in SOCS3 expression in treated and control sample! ΔΔ Ct method: Example

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no need for dilution series  less material needed, faster BUT: amplification efficiency of target and endogenous control must be comparable ΔΔ Ct method

Efficiency of amplification:

Efficiency of amplification Changes in efficiency change the slope when you use the  logarithmic scale. ΔΔ Ct method

Validation of efficiency:

Validation of efficiency y = - 3.3276x + 27.712 Effic = 100 % C t Value 0 2 4 6 8 1 0 Log [Input mRNA] 10 15 20 25 30 35 y = - 3.3683x + 36.009 Effic = 98 % D Target Endogenous control equal efficiency or equal slopes for target and endogenous control - Acceptible slope = 3.2 - 3.8 (Efficiency 83 – 105 %) y = - 4. 586x + 24.889 Effic = 67 % Target

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Does the target have a similar amplification efficiency to the endogenous control? NO YES Standard Curves Ct method ΔΔ Ct method

Primers and probe design:

Primers and probe design

Primers and probe design:

Primers and probe design Primer Tm 58 - 60ºC 20 - 80% GC Length 9 - 40 <2ºC difference in Tm between the two primers Maximum of 2 G or C at 3’ end Amplicon 50 - 150 bp in length As close to the probe as possible without overlapping Probe Tm 10ºC higher than Primer Tm (7ºC for Allelic Discrimination) 20 - 80% GC Length 9 - 40 bases No G on the 5’end <4 contiguous G’s Must not have more G’s than C’s

Primers and probe design:

Primers and probe design Theoretical Tms may not always be accurate This would lead to an imbalance between the two primers  Primer optimisation

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Primer Concentration Optimization MATRIX FORWARD REVERSE 50nM 300nM 900nM 50nM 300nM 900nM 50 / 50 50 / 300 50 / 900 300 / 50 900 / 50 300 / 300 300 / 900 900 / 300 900 / 900 Primers and probe design

Primer Optimisation for SYBR Green I:

Primer Optimisation for SYBR Green I Perform 50/300/900nM primer matrix: Choose the optimal primer concentration Lowest Ct Highest Rn No amplification in negative control Probe Optimisation (Taqman) Increase probe concentration from 50nM to 300nM Lowest Ct without excess probe

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Primers and probes for both target gene (SOCS3) and reference gene (18S) in the same tube Primers for reference gene (18S) must be limited 18 S target gene Multiplex reactions

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Real time detection: Quantitation of gene expression Quantitation of RNA, DNA, cDNA Viral quantitation … Endpoint detection: Allelic discrimination (SNP genotyping) Plus/minus studies Pathogen detection … APPLICATIONS

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Assays +/- Alelic Discrimination (SNPs) Absolute Quantitation Relative Quantitation End Point Real Time Absolute Quantitation Relative Quantitation Assays +/- Allelic Discrimination (SNPs) ALLELIC DISCRIMINATION End point detection

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Allele 1 Mismatch Mismatch FAM VIC VIC FAM Allele 2     Tamra ™ Tamra Tamra Tamra Perfect match Perfect match FAM ™ -labelled probe is specific for Allele 1 VIC ™ -labelled probe is specific for Allele 2 ALLELIC DISCRIMINATION Principle: 2 primers, 2 probes

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relies on competition between the two probes Tm of the mismatched probe < Tm of perfectly matched probe Annealing/extension temperature of 60°C allows binding and cleavage of correct probe and destabilisation of incorrect probe Allele 1 Incorrect Probe FAM ™ VIC ™   Tamra Tamra ™ Correct Probe Tm = 65ºC Tm = 55ºC Allele 1 ALLELIC DISCRIMINATION Mechanism

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Allele 1 Allele 1&2 Allele 2 ALLELIC DISCRIMINATION Typical output FAM VIC FAM VIC VIC FAM  homozygote for allele 1  homozygote for allele 2  heterozygote

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ALLELIC DISCRIMINATION Typical output

Practical tips:

Practical tips Prevention of contamination (gloves, filtered tips, pre- and post-PCR area) Precise pipetting; Use triplicates Include positive and negative controls For gene expression quant. use intron-spanning primers to avoid genomic contamination or use DNAse treatment after RNA purification genomic DNA cDNA EXON 1 EXON 2 EXON 1 EXON 2

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