iq5 & MYQ Real-Time PCR
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1 iq5 & MYQ Real-Time PCR
2 Why Real Time PCR? More accurate than RT PCR Publications are requiring real time PCR data rather than RT PCRs Ease and speed of quantification Can do many technical replicates without exhausting cdna template
3 Real-Time PCR: Applications Real-Time qpcr provides information for relative or absolute measurements of starting material. Gene Expression Studies Chromatin Immunoprecipitation (ChIP) Microarray Validation Transgene Analysis/GMO Testing Viral/Bacterial Load Studies Allelic Discrimination/SNP
4 Limitations of Conventional PCR Amplification is exponential initially In theory, the amount of DNA produced at every cycle should double, Product = Template x 2 n (n = # of cycles) Log Target DNA Theoretical Observed But the exponential phase becomes linear as reactants become rate-limiting Cycle #
5 RT PCR is an End-Point Analysis 96 identical reactions will have very different final amounts of fluorescence at endpoint due to differences in kinetics and pipetting
6 Quantitative PCR Through the use of fluorescent dyes that detect PCR products, real-time PCR measures the reaction directly during amplification
7 iq5 Optical System
8 iq5 Utilizes Filter Wheels Emission Filter Detector Excitation Filter Light Source (Simple halogen bulb)
9 Emission Filter Windows 490/ / / Cy5 FAM ROX iq5 detects up to 5 dyes colors - MYQ detects SYBR Green only
10 Detection of quantitative Data Theoretical Observed Log Target DNA Detector Cycle #
11 Intercalation Dyes: SYBR Green I Displays strong fluorescence in the presence of dsdna 3 5 ID ID ID Taq 5 Taq ID ID 5 3
12 Cleavage-based assay: TaqMan TM Add iq Supermix, Hybridization Probe and sample Primers 3 5 d. NTPs Thermal Stable DNA Polymerase R Probe 5 3 Q 5 3 Denaturation 3 Taq R Q Annealing
13 Cleavage-based assay: TaqMan TM 3 Q R Extension Step 3 Q R Taq 5 3 R 5 3 Q Taq Taq Q R Taq 3 Q R
14 Threshold Cycle (C T ) - Assigned point at which the fluorescence rises appreciably above background -The C T can be placed anywhere in the exponential (log-linear) phase C T
15 Results are More Accurate in the Log Phase 96 identical reactions will have nearly identical C T values when measured at the linear phase of amplification C T
16 Threshold Cycle (C T ) - At the CT, the fluorescence correlates well with the starting copy number - BioRad claims a dynamic range of 9 orders of magnitude - Each 10x dilution equals 3.3 cycles 2 n = 10 fold n(ln2) = ln10 n = ln10 ln 2 n = 3.32
17 Standard Dilution Series 1) To perform absolute quantification 2) To examine the efficiency of the qpcr reaction (This can influence the results of your experiment significantly)
18 C T Values Determine the Copy Number Standard Dilution Series can be used to determine the Copy Number or Concentration of an Unknown
19 Example of Good Efficiency Correlation Coefficient >0.9 Efficiency >90%
20 Example of Unreasonably High Efficiency How do you get an efficiency of >200%?
21 Real-time PCR Efficiencies Efficiency =10-1/slope % Efficiency=10-1/slope -1 X % 90% Primer-dimer! Pipette calibration Dilution Errors Target Range Secondary structures Pipette calibration Dilution errors
22 Primer Dimers Raise the Apparent Efficiency Optimization of RT-qPCR: Annealing Temperature A A B A B A B A B A B A B A B A B Melt curve dimers Primer Conc. 200 nm B = NTC A = Template + primers B = Template only (NO primers) data from Walling lab
23 What Are Primer Dimers?
24 Primer Concentration Affects qpcr Efficiency Optimization of RT-qPCR: Primer Concentration 1 2 NTC 1: 100 nm 2: 200 nm data from Walling lab
25 Primer Concentration Primer-dimers can be influenced by template concentration 10,000 copies template 2,000 copies 400 copies Dimers Amplicon No template control Dimers only
26 Genomic DNA Contamination Can Affect PCR Efficiency Optimization of RT-qPCR: Genomic DNA Contamination Genomic DNA cdna product data from Walling lab - DNAse treatment will reduce contamination - Also design primers across intron/exon boundaries
27 Melt Curve Analysis Analysis at the end of the qpcr run to determine the melting temperature (T m ) of PCR products T m of amplicon Plot rate of change of fluorescence vs. temperature
28 Why Do Melt Curve? Identification of non-specific products Mutation detection/allelic discrimination
29 Primer Dimers Raise the Apparent Efficiency Optimization of RT-qPCR: Annealing Temperature A A B A B A B A B A B A B A B A B Melt curve Primer Conc. 200 nm B = NTC A = Template + primers B = Template only (NO primers) data from Walling lab
30 iq5 Features for Reaction Optimization Continuous Data Collection Thermal Gradient Melt Curve
31 Continuous Data Collection icycler iq features a continuous data collection that is designed to focus analytical scrutiny on subsets of the data gathered Optimize annealing/extension time Chemistry kinetics
32 Optimizing PCR Reactions: 2-Step PCR vs 3-Step 95ºC 58ºC 72ºC Denaturation Annealing Extension In some cases, can eliminate extension step since all amplification has already occurred during annealing
33 iq5/myq Thermal Gradient Used for annealing/extension temperature optimization for PCR reaction specificity and efficiency Up to 24 o C gradient range programmable across block.
34 iq5/myq Thermal Gradient Look for specific product formation without primer dimers Dilution series of primer [ ] Temperature Gradient (annealing temperature)
35 How do I get started?
36 Workflow RNA preps (Qiagen) cdna prep (1 g starting RNA) using 2-step kits - Ambien Retroscript for RT - In Vitrogen Superscript III - BioRad iscript RT Dilute cdna template (~1:10) qpcr (BioRad SYBRGreen SuperMix) Data analysis
37 Reaction Set-Up qpcr reaction (per reaction) Master mix SYBRGreen SuperMix (Biorad) Primer forward (10uM) Primer reverse (10uM) Water 12.5ul of 2x 0.5ul (200nM) (can use 100 to 500nM) 0.5ul 9.5ul 23ul Template 2ul (typically 1:10 dilution cdna) Total volume 25ul Set-up can be at room temperature if using the BioRad SuperMix other hot-start Taq polymerase
38 Plate Set-Up Reference gene GOI 1 GOI 2 GOI 3 Master mixes for each primer set Condition 1 Condition 8 Set-up can be at room temperature if using the BioRad SuperMix
39 qpcr Optimization A. Optimize conditions: 1) Annealing temperature 2) Primer concentration 3) Annealing/extension time 4) Sample prep protocol B. Test efficiency of primers: 1) Primer-dimer 2) Secondary structure SYBR Green I C. Set up SYBR Green I experiments: 1) Standards and unknowns
40 Template DNA Genomic DNA - Cut with restriction enzyme that does not cut within amplified region - Boil DNA stock for 10 min and then onto ice Plasmid DNA - If it doesn t work, linearize plasmid with restriction enzyme that does not cut within amplified region cdna - Treat total RNA with RNase-free DNase prior to reverse transcription - Try to design primers at exon boundaries to avoid genomic DNA amplification - Use enzyme that has RNaseH activity to digest away RNA from RNA:DNA hybrid after making cdna (kits are designed with this feature)
41 Primer Design There is nothing special about qpcr primers, but there are some considerations: - Optimal Amplicon length bp (can be longer) - When designing primers, check for - predicted secondary structure - predicted primer-dimer formation - can also check for predicted hairpins in the amplicon - Free tools at - IDT website (Google IDT) - mfold website - Premier Biosoft - When designing multiple primers to be run on the same plate try to design primers with similar T m
42 Amplicon Secondary Structure Reverse primer A Forward Primer Reverse Primer A Primer efficiency = 66.3%
43 Amplicon Secondary Structure Reverse Primer B Reverse primer B Forward Primer Primer efficiency = 95.8 %
44 Getting Consistent Results Do not underestimate the importance of using: Dedicated set of pipettors for real-time PCR Screwcap tubes Aerosol-barrier filter tips Hot-start Taq polymerase (antibody mediated) PCR-grade water Pipet carefully!
45 Getting Consistent Results Wear gloves Create master mixes to average out error Mix very well by vortexing (at least 5 secs) No-template control to check for contamination Prepare reactions in replicate ideally triplicate Seal the tape well press firmly Occasionally inspect reaction module for salt or dirt buildup Centrifuge to eliminate bubbles (bottom of well)
46 Data Analysis Relative Quantification Absolute Quantification Allelic Discrimination
47 Calculating Relative Expression Pfaffl method (Efficiencies are normalized) Tissue #1: Tissue #2: Reference Primers (C T ) GOI Primers (C T ) (From Standard curve) Efficiency: 90% = % = 2 Delta C T : = = 2 Fold induction in tissue #1 relative to tissue #2 = 2 target deltact target (24-22 = 2) 1.9 reference deltact reference (20-21 = -1) = 4 = All samples should be in triplicate with at least two biological replicates
48 Calculating Relative Expression Pfaffl method: (efficiencies are normalized) Mutant or treated: Reference Primers (C T ) 21 GOI Primers (C T ) 22 Wild type or untreated: (From Standard curve) Efficiency: 90% = % = 2 Delta C T : = = 2 Fold induction in treated relative to untreated = 2 target deltact target (24-22 = 2) 1.9 reference deltact reference (20-21 = -1) = 4 = All samples should be in triplicate with at least two biological replicates
49 Displaying Results Can export results to Excel within the iq5 software Well Fluor Type Identifier Replicate # Threshold Cycle (Ct) Ct Mean Ct Std. Dev 30 mins A01 SYBR Unkn 1A_DMSO30_Ub A02 SYBR Unkn 1A_DMSO30_Ub A03 SYBR Unkn 1A_DMSO30_Ub A04 SYBR Unkn 1B_DMSO30_BL A05 SYBR Unkn 1B_DMSO30_BL A06 SYBR Unkn 1B_DMSO30_BL A07 SYBR Unkn 2A_Pru30_Ub A08 SYBR Unkn 2A_Pru30_Ub A09 SYBR Unkn 2A_Pru30_Ub A10 SYBR Unkn 2B_PrU30_BL A11 SYBR Unkn 2B_PrU30_BL A12 SYBR Unkn 2B_PrU30_BL B01 SYBR Unkn 3A_BL30_Ub B02 SYBR Unkn 3A_BL30_Ub B03 SYBR Unkn 3A_BL30_Ub B04 SYBR Unkn 3B_BL30_BL B05 SYBR Unkn 3B_BL30_BL B06 SYBR Unkn 3B_BL30_BL B07 SYBR Unkn 4A_NAA30_Ub B08 SYBR Unkn 4A_NAA30_Ub B09 SYBR Unkn 4A_NAA30_Ub B10 SYBR Unkn 4B_NAA30_BL B11 SYBR Unkn 4B_NAA30_BL B12 SYBR Unkn 4B_NAA30_BL C01 SYBR Unkn 5A_BLPrU30_Ub C02 SYBR Unkn 5A_BLPrU30_Ub C03 SYBR Unkn 5A_BLPrU30_Ub C04 SYBR Unkn 5B_BLPrU30_BL C05 SYBR Unkn 5B_BLPrU30_BL C06 SYBR Unkn 5B_BLPrU30_BL C07 SYBR Unkn 6A_NAAPrU30_Ub C08 SYBR Unkn 6A_NAAPrU30_Ub C09 SYBR Unkn 6A_NAAPrU30_Ub C10 SYBR Unkn 6B_NAAPrU30_BL C11 SYBR Unkn 6B_NAAPrU30_BL C12 SYBR Unkn 6B_NAAPrU30_BL BioRad iq5
50 Displaying Results RT-PCR Quantitative-PCR DMSO ES1 BL NAA BL/ES1 NAA/ES1 Fold Induction BL-responsive gene at5g hrs 0 ES 3hr BL 3hr BL/ES 3hr NAA 3hr NAA/ES 3hr - Comparison of chemical vs non-treated samples - Histogram generated in Excel BioRad iq5
51 Data Analysis FINIS
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