MIQE Guidelines. Simply - fluorescent molecules are used to monitor the reaction while amplification is taking place.

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Central Dogma MIQE Guidelines Minimum Information for Publication of Quantitative Real- Time PCR Experiments Central dogma describes information flow from DNA RNA protein

two primers synthesizes using allows 94 thermostable DNA o targeting C DNA complementary polymerase of specific sequences to template in 5` to 3` direction Primers are

of PCR doubles the number of progeny DNA duplexes (which can then act as template as well) 94 1 cycle = 2 1 copies of o C starting template 50 65 o C 25 cycles = 2 25

You are able to view this occurring in real-time on your instrument.

1 Central Dogma MIQE Guidelines Minimum Information for Publication of Quantitative Real- Time PCR Experiments Central dogma describes information flow from DNA RNA protein Frank Lin Product Specialist Gene Expression Division Bio-Rad Laboratories Taiwan DNA Structure Polymerase Chain Reaction The Targeted use polymerase DNA of replication two primers synthesizes using allows 94 thermostable DNA o targeting C DNA complementary polymerase of specific sequences to template in 5` to 3` direction Primers are complementary to opposite o C strands of target region but not complementary to any other sequences EXTEND ANNEAL DENATURE STRANDS PRIMERS 72 o C 72 o C Each cycle of PCR doubles the number of progeny DNA duplexes (which can then act as template as well) 94 1 cycle = 2 1 copies of o C starting template o C 25 cycles = 2 25 copies of starting template What is Real-Time PCR? Reality vs. Theory Simply - fluorescent molecules are used to monitor the reaction while amplification is taking place. You are able to view this occurring in real-time on your instrument. Amplification is exponential, but the exponential increase is limited: l A linear increase follows exponential Eventually plateaus Real-Time PCR allows us to see the exponential phase so we can calculate how much we started with. Log Target DNA Theoretical Real Life Cycle # 1

Quantitative PCR (qpcr) Real Time PCR Real-time qpcr enables assessment of the reaction after each cycle 100 Product Amount Conventional PCR

appreciably above background 96 replicates of an identical reaction can have very different final amounts of fluorescence Threshold Cycle, C t C T

concentration Correlates strongly with the starting copy number If you have twice the template, you get to C t one cycle earlier If you have half

2 Quantitative PCR (qpcr) Real Time PCR Real-time qpcr enables assessment of the reaction after each cycle 100 Product Amount Conventional PCR Threshold 0 Cycle number 35 Baseline Log phase Plateau Threshold Cycle, C T End Point Measurements The point at which the fluorescence rises appreciably above background 96 replicates of an identical reaction can have very different final amounts of fluorescence Threshold Cycle, C t C T value v.s. concentration Correlates strongly with the starting copy number If you have twice the template, you get to C t one cycle earlier If you have half the template, you reach C t one cycle later Is linear with the log of starting copy number over six or more orders Human genomic DNA with SYBR 1 cycle = 2 fold difference 3.32 cycles 10 fold difference Assumes 100% efficiency Y= N0 2 n Y=N 0 (1+E) n ng of template- FV Leiden primers 2

3 Quantitative PCR (qpcr) C T is linear with the log of starting copy number (standard curve) Create a standard curve with 10-fold serial dilutions of PCR product assign arbitrary values Compare values from standards with values for unknown sample 100 Product Amount STD 1: 1,000,000 STD 2: 100,000 STD 3: 10,000 STD 4: 1,000 STD 5: 100 STD 6: 10 Sample: 6,592 0 Cycle number 35 r = is The a measure slope of of the how standard well the curve actual can be data directly fit to correlated the standard to the curve. = (explained efficiency variation/total of the reactions: variation) Efficiency (E) = [10 (-1/slope) ] - 1 Aim for when R value slope (Correlation = -3.32, Efficiency Coefficient) = 100% of 0.98 E Slope Slope = - [1/log (1+E)] log (1+E) = - (1/slope) E = 10 [-1/slope] - 1 Aim for Efficiency Values: Good = % Fantastic = % What are the most common detection strategies used for Real-Time PCR? Real-Time PCR DNA binding dyes These fluorescent molecules can be used Non-specific DNA binding dyes SYBR Green I Ethidium Bromide Add Master Mix & Sample Primers Reaction Tube d. NTPs Thermal Stable DNA Polymerase DNA binding dyes Specific Hybridization Probes/Primers Man molecular beacons dual-oligo FRET pairs Scorpions /Amplifluor /LUX Denaturation l BD Annealing 3

4 DNA binding dyes Melt Curve Analysis Fluorescence decrease as the temperature increase: Extension BD BD 1. DNA strands start to separate 2. SYBR green looses its binding to the DNA 3. Fluorescence rapidly decreases BD BD BD Extension Continued Apply Excitation Wavelength l l l BD BD BD BD BD l l Repeat Melt Curve Analysis Cleavage-based assay: Man Probe -The melting temperature of the amplicon can easily be detected. -Contaminating DNA, primer dimer or false priming is seen as an additional peak. Add Master Mix & Sample Primers Reaction Tube R d. NTPs Thermal Stable DNA Polymerase Probe Q Denaturation l R Q Annealing Cleavage-based assay: Man Probe Extension Step Q R 1. Strand Displacement Q 2. Cleavage R Q R What data do you perform now? 3. Polymerization Complete 4. Detection Q Q R l R 4

Historically before qpcr After the arrival of qpcr Laborious

10 fold! Northern I don t Blotssee this Southern as 10 Blots fold.

Competitive PCR Assay development was much easier.

Results were achieved much faster. Data sets grew larger.

Instruments generated numerical outputs.

experiments has resulted in many publications containing

5 Historically before qpcr After the arrival of qpcr Laborious techniques were used to examine specific gene expression levels; 10 fold! Northern I don t Blotssee this Southern as 10 Blots fold. It looks more like RNAse Protection Assays three fold to me! Competitive PCR Assay development was much easier. Sample required was much smaller. Results were achieved much faster. Data sets grew larger. Etc etc These methods were Simiquantitative at best. Instruments generated numerical outputs. The Problem QPCR papers Perceived simplicity of running qpcr experiments has resulted in many publications containing seriously misleading results. Key word search with Real-time PCR in NCBI Pubmed Questionable Data Retracted Paper Slope : Eff = % Slope : Eff = % 5

Autism and MMR Vaccine Autism and MMR Vaccine Lingering Effects of Bad Data What can be done?

investigations: the MIBBI project. Nature biotechnology, volume 26, number 8, August 2008, 889-896.

Lefever S, Hellemans J, Pattyn F, Przybylski DR, Taylor C, Geurts R, Untergasser A, Vandesompele J; on behalf of the

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments.

6 Autism and MMR Vaccine Autism and MMR Vaccine Lingering Effects of Bad Data What can be done? MIBBI,RDML and MIQE A new beginning Promoting coherent minimum reporting guidelines for biological and biomedical investigations: the MIBBI project. Nature biotechnology, volume 26, number 8, August 2008, RDML: structured language and reporting guidelines for real-time quantitative PCR data. Lefever S, Hellemans J, Pattyn F, Przybylski DR, Taylor C, Geurts R, Untergasser A, Vandesompele J; on behalf of the RDML consortium. Nucleic Acids Res Feb 17. The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. Clin Chem Feb Essential information (E) must be submitted with the manuscript 28 Desirable information (D) should be submitted if available 6

desired information in litterature; Experimental Design Sample Information Nucleic Acid

qpcr Validation Data Analysis Experimental Replicates Sample Information NTC Sample

plant tissues ) Sampling techniques (biopsy materials, single-cell sampling, laser

7 What is MIQE? It s a Checklist Experimental Design qpcr community driven guidelines for essential and desired information in litterature; Experimental Design Sample Information Nucleic Acid Extraction Reverse Transcription qpcr Target Information qpcr Oligonucleotides qpcr Protocol qpcr Validation Data Analysis Experimental Replicates Sample Information NTC Sample Information Acquisition Sample Information Processing and Storage Source (soil, blood, urine, plant tissues ) Sampling techniques (biopsy materials, single-cell sampling, laser microdissection ) Description (percentage of the biopsy made up of tumor cells) Mass/Volume Homogeneity Processing procedure Storage (-80C vs -20C; duration ) Prep time to extraction (-80C) Sample degradation 7

Nucleic Acid Extraction Sample Extraction DNA / RNA

RNA Kits Contaminants Starches Lipids Metals

Sample degradation Inhibitors copurified Effects of

restriction enzyme that does not cut within amplicon

it doesn t work, linearize plasmid with cdna Prior

enzyme that has RNaseH activity to digest away RNA

8 Nucleic Acid Extraction Sample Extraction DNA / RNA Aquapure TM Genomic DNA Isolation Kit Aurum TM Total RNA Kits Contaminants Starches Lipids Metals Extraction contaminants Phenol chloroform Salts Sample degradation Inhibitors copurified Effects of EDTA Template DNA Preparation Genomic DNA Cut with restriction enzyme that does not cut within amplicon Boil DNA for 10 min and then onto ice Plasmid DNA If it doesn t work, linearize plasmid with restriction enzyme that does not cut within amplicon cdna Prior to RT: Treat RNA with RNase-free DNase After RT: use enzyme that has RNaseH activity to digest away RNA from RNA:DNA hybrid 8

6 0 0 0 4 0 0 0 3 0 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0

0 Sample and Template Preparation Analysis of RNA purity and integrity

Assay - Quantification NanoDrop Quantification and purity Experion -

4 5 6 7 8 Sample Corresponding Nanodrop Readings Conc (ng/ul) A260/280*

40 5 min @ 90C 115 2.06 2.37 10 min @ 90C 115 2.03 2.

00 2.32 Samples 6,7,8 are highly degraded. 4 hr @ 90C 118 1.89 2.

9 Sample and Template Preparation Analysis of RNA purity and integrity Extract and analyze RNA Careful quantification is necessary RiboGreen Assay - Quantification NanoDrop Quantification and purity Experion - Quality and quantification Experion Virtual Gel L C h 2h 4h L Sample Corresponding Nanodrop Readings Conc (ng/ul) A260/280* A260/230* Control-no heat C C C C C C Samples 6,7,8 are highly degraded. 4 90C *Generally accepted ratios (A260/280 and A260/230) for good quality RNA are > 1.8. RQI Value & Color Coded Classification Experion Analysis of RNA Total RNA analysis Experiment: Evaluate sirna-mediated gene silencing Prevent faulty conclusions GAPDH sirna A Scrambled sirna control B C T 6.8 C T % 89% Cq of and transcripts Reverse Transcription 9

cdna Synthesis RT protocols: Experiment: Testing Results Across a Range of cdna Input Concentrations iscript qrt-pcr Standard Curve Comparison: cdna serial dilution vs.

6% RNA isolated from HeLa cells d u 5 1 2 3 4 5 6 7 8 9 Log Starting Quantity (femtograms of input RNA) Note: 1/10th of cdna reaction used for PCR Reverse transcription Strategies Increase primer

10 cdna Synthesis RT protocols: Experiment: Testing Results Across a Range of cdna Input Concentrations iscript qrt-pcr Standard Curve Comparison: cdna serial dilution vs. total RNA serial dilution β-actin cdna Standard Total RNA Standard T e cdna total Slope Corr. Coef Intercept PCR efficiency 97.1% 97.6% RNA isolated from HeLa cells d u Log Starting Quantity (femtograms of input RNA) Note: 1/10th of cdna reaction used for PCR Reverse transcription Strategies Increase primer design flexibility & prevent bias optimum blend of oligo(dt) & random primers AAAAA Oligo dt Random primers Oligo dt and Random primers Gene specific primers RNase H RNase inhibitors RT 100, 10, 1, and 0.1 ng input RNA Primer pairs for 5 end (green trace - ~60 bp) Primer pairs for 3 end (orange trace - ~70 bp) Obtain Accurate Results potent blend of RNase inhibitor iscript works with a broad range of RNA Notice the Ct delay Range of iscript is100fg 1µg of input RNA Competitors cannot reliably achieve this range, and sometimes require two RTs for low and high amounts of input RNA RT with iscript (green trace) RT with iscript + RNaseA (red trace) RT with iscript without RNase inhibitor + RNase (black trace) µg, 10-6 ng, 10-9 pg, fg, iscript Competitors 10

11 qpcr Target Information Bio-Rad s iscript cdna Synthesis Kit Invitrogen s SuperScriptII First-Strand Synthesis System for RT-PCR 13 tubes! NCBI Blast analysis Free resource Blast sequence Stack sequence gov/blast/blast.cgi Amplicon Secondary Structures Effect of primer location Primer A: η = 66.3 % Primer B: η = 95.8 % Primer A Bad location for primers Good location for primers Reverse Primer B Primer B Forward Primer Reverse Primer A

12 qpcr Oligonucleotides Primer Design Designs Primers Designs Internal Oligos Provides multiple outputs Free Web software provided by Steve Rozen and Whitehead Institute for Biomedical Research. Beacon Designer RTPrimerDB qpcr Protocol 12

Recipe, Protocol, Consumable, and Instrument 1 RXN # RXNs Number of RXNs 1

3 um) 0.6 54.0 10uM IL1β246R (-->0.3 um) 0.6 54.0 10uM FAM-IL1β BHline (--> 0.

3 um) 0.6 54.0 100uM HEX-GAPDHBHLine (--> 0.2uM) 0.4 36.0 1440.

SsoFast EvaGreen Supermix Sso7d from Sulfolobus solfataricus 7kD, 63 aa.

Binds to dsdna (3-6 bp/protein molecule) Monomeric High Speed High

Higher activity Tolerant to PCR inhibitors Sso7d-fusion enzymes Enhanced

Sso7d- Processivity 15-19nt 96-109nt 2-3nt 35-39nt 2-4nt 26-39nt 1x 0.

13 Recipe, Protocol, Consumable, and Instrument 1 RXN # RXNs Number of RXNs 1 90 TE (1X) X iq PowerMix (-->1X) uM IL1β5101F (-->0.3 um) uM IL1β246R (-->0.3 um) uM FAM-IL1β BHline (--> 0.2uM) uM GAPDH 169F (-->0.3 um) uM GAPDH 290R (-->0.3 um) uM HEX-GAPDHBHLine (--> 0.2uM) Total Template 4 3rd Generation Reagents 3rd Generation Reagents: SsoFast EvaGreen Supermix Sso7d from Sulfolobus solfataricus 7kD, 63 aa. SsoFast TM Series Supermix Thermostable (Tm >90 C) No sequence preference Binds to dsdna (3-6 bp/protein molecule) Monomeric High Speed High Tolerance High Dynamic Range Minimal inhibition of PCR by use of EvaGreen Higher activity Tolerant to PCR inhibitors Sso7d-fusion enzymes Enhanced processivity PCR Inhibition caused by Dye Enzyme Sso7d- Pfu Pfu-Sso7d Sso7d- Processivity 15-19nt nt 2-3nt 35-39nt 2-4nt 26-39nt 1x 0.75x 0.5x 0.25x 1.5x 2x 5x 0.5x Processivity is enhanced 5 to 10-fold Source: Barrett et al., Quantace Ltd. 13

Even Dye Redistribution EvaGreen 1.2kb 200bp 2.5kb 200bp 1.2kb 2.5kb Source: Barrett et al.

The even dye redistribution gives equal signal intensities from different sized DNA

95 o C 10 sec 60 o C 60 sec / melt CCl26 amplified using Other Reagent A: 5ul Assay 95 o

SsoFast EVAGreen Supermix: 5ul Assay 98 o C 30sec / 50x95 o C 1 sec 60 o C 5 sec / melt

o C 30 sec / melt analysis CCl26 amplified using Other Reagent C: 5ul Assay 95 o C 20sec

50x 95oC 10 sec 60oC 60 sec / melt CCl26 amplified using Other Reagent A: 5ul Assay 95 o

14 Even Dye Redistribution EvaGreen 1.2kb 200bp 2.5kb 200bp 1.2kb 2.5kb Source: Barrett et al., Quantace Ltd. The even dye redistribution gives equal signal intensities from different sized DNA fragments Phenol Chloroform (extraction mix) Phenol Chloroform (extraction mix) <0.78% <0.39% <1.56% 1.56% 0.78% 3.125% CCl26 amplified using Bio-Rad iq SYBR Green Supermix: 5ul Assay95 o C 3 min / 50x 95 o C 10 sec 60 o C 60 sec / melt CCl26 amplified using Other Reagent A: 5ul Assay 95 o C 5min / 50x 95 o C 15 sec 60 o C 60 sec / melt analysis CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98 o C 30sec / 50x95 o C 1 sec 60 o C 5 sec / melt analysis <0.78% 1.56% <0.78% 1.56% CCl26 amplified using Other Reagent B: 5ul Assay 95 o C 20sec / 50x 95 o C 3 sec 60 o C 30 sec / melt analysis CCl26 amplified using Other Reagent C: 5ul Assay 95 o C 20sec / 50x 95 o C 3 sec 60 o C 30 sec / melt analysis Blood Serum Blood Serum < % % <0.0089% 0.039% <2.5 % 10 % CCl26 amplified using Bio-Rad iq SYBR Green Supermix: 5ul Assay 95oC 3 min / 50x 95oC 10 sec 60oC 60 sec / melt CCl26 amplified using Other Reagent A: 5ul Assay 95 o C 5min / 50x 95 o C 15 sec 60 o C 60 sec / melt analysis CCl26 amplified using Bio-Rad SsoFast EVAGreen Supermix: 5ul Assay 98 o C 30sec / 50x95 o C 1 sec 60 o C 5 sec / melt analysis <0.0089% 0.039% <0.0089% 0.039% CCl26 amplified using Other Reagent B: 5ul Assay 95 o C 20sec / 50x 95 o C 3 sec 60 o C 30 sec / melt analysis CCl26 amplified using Other Reagent C: 5ul Assay 95 o C 20sec / 50x 95 o C 3 sec 60 o C 30 sec / melt analysis 14

Inhibitor Concentration Not Having Any Significant Effect on qpcr Reaction High Dynamic Range SsoFast EvaGreen Supermix ABI Fast SYBR Green Mastermix Inhibitor Max Tested DilutionSer ies EVA iq SYBR

25 mm 125 mm 62.5 mm 31.25 mm NaAc 250 mm 2 fold 62.5 mm 62.5 mm 62.5 mm 62.5 mm 15.63 mm Phenol/Chloroform 6.25 % 2 fold 1.53 % 0.781 % 0.781 % 0.781 % 0.391 % Efficiency = 104.0%, r = 0.

15 Inhibitor Concentration Not Having Any Significant Effect on qpcr Reaction High Dynamic Range SsoFast EvaGreen Supermix ABI Fast SYBR Green Mastermix Inhibitor Max Tested DilutionSer ies EVA iq SYBR ABI INV Euro Ethanol Isopropanol 25 % 17.5 % 2 fold 2 fold 12.5 % 8.75 % 6.25 % % % 2.18 % % % % % Fast Protocol 36 min KCl 500 mm 2 fold 62.5 mm mm 125 mm 62.5 mm mm NaAc 250 mm 2 fold 62.5 mm 62.5 mm 62.5 mm 62.5 mm mm Phenol/Chloroform 6.25 % 2 fold 1.53 % % % % % Efficiency = 104.0%, r = Efficiency = 110.4%, r = Blood Serum 10 % 4 fold 2.5 % % % % % Invitrogen EXPRESS SYBR GreenER Qiagen QuantiFast SYBR Mix Total Blood (+hep) 10 % 4 fold % % % % % Heparin U 2 fold U U U U U Soil Coffee 25 % 25 % 4 fold 4 fold 6.25 % 6.25 % 1.56 % 1.56 % 1.56 % 1.56 % 6.25 % 1.56 % 0.39 % 1.56 % Fast Protocol 36 min Efficiency = 89.3%, r = Efficiency = 98.5%, r = Bio-Rad Mix in Green Competitor Mix in Blue qpcr Validation How to choose Optimization the annealing temperature? Evidence of optimization - thermal gradient Gradient optimization dynamic thermal gradient 10 o C Above Single Plate Optimisation of Annealing Temperature and PCR Efficiency via Slope of Calibration Curve Thermal gradient Dilution series o C Below 15

16 SYBR Green Validation Gradient Optimization of Annealing Temperature Use a serial dilution of template to test primers across a broad dynamic range. 67 o C Efficiency = 68% Include representative unknown samples. Evaluate specificity, efficiency, reproducibility and dynamic range. Gel checking. Serial dilutions at 8 temps from 55 o C to 68 o C Reactions at 62 o C annealing have low Cts and highest reaction efficiency 62 o C Efficiency = 99% Efficiency = 98% 56 o C Data Analysis qpcr Program/Software CFX Manager Absolute Quantization Relative Quantization 16

Allelic Discrimination End Point Analysis HRM mutation analysis Gene Expression Normalization - Comparative C T Lung cancer screen in

normalization Normalized Expression ( CT) Accounts for loading differences Usually normalize to reference gene Relative quantity of

International Consortium CT Assume 100% efficiency Only one Ref Gene Pfaffl Modification Accounts for efficiency differences Only one

developer group, a member community and supporters 41 supporters and members from 20 different countries 22 academic Austria,

17 Allelic Discrimination End Point Analysis HRM mutation analysis Gene Expression Normalization - Comparative C T Lung cancer screen in KRAS mutation on CFX96 (Dr Chen in SSH) SsoFast EvaGreen Supermix Protocol 98 o C 2 mins (40 cycles) 98 o C 5 sec 61 o C 10 sec (Melt) 95 o C 30 sec 75 o C 5 sec (+0.2 o C/ cycle to 95 o C) Relative Quantity ( CT ) Not normalized Normalization accomplished via equal loading of samples Post analysis normalization Normalized Expression ( CT) Accounts for loading differences Usually normalize to reference gene Relative quantity of GOI is normalized by the relative quantity of the reference genes Gene Expression Normalization - Normalized Expression The RDML International Consortium CT Assume 100% efficiency Only one Ref Gene Pfaffl Modification Accounts for efficiency differences Only one Ref Gene Vandesompele Method Accounts for efficiency differences Allows multiple reference genes for normalization Simple Complex Key developer group, a member community and supporters 41 supporters and members from 20 different countries 22 academic Austria, Switzerland, France Spain, Israel, Canada,... Sweden (2) Germany (3) UK (3) USA (5) 19 companies USA (6) UK (2) Belgium (2) Portugal, Austria Switzerland, Israel, Australia, Korea,... Goal: develop and maintain the RDML data exchange format 17

silentfect Transfectin Gene Pulser Xcell

SmartSpec Plus Reverse Transcription and

18 Conforming to MIQE: an example Complete solutions RQI value The Bio-Rad technote 5859 provides an excellent summary of a MIQE compliant experiment in practice, from start to finish Gene Modulation RNA Purification Aurum Total RNA Kits silentfect Transfectin Gene Pulser Xcell iscript cdna kits iq Multiplex Powermix MJ Mini iscript Cycler reagents SsoFast reagents MiniOpticon, MyiQ2 & CFX96 Profiling & Quantification RNA Analysis (Quality and Yield) Experion System SmartSpec Plus Reverse Transcription and Amplification Gradient function Multiple ref genes &efficiency qbase software Beacon Designer Questions? 18

Q-PCR theory and points for attention

Central Dogma Q-PCR theory and points for attention Central dogma describes information flow from DNA RNA protein 產品專員 / 陳立德 DNA Structure Polymerase Chain Reaction Targeted The polymerase use DNA of replication