Rotor-Gene Q Pure Detection Designed to drive your research forward - 1 -
What is real-time PCR? Real-time PCR allows monitoring of the newly generated PCR product during amplification with the help of fluorescent dyes Synonyms: Kinetic PCR Online PCR TaqMan PCR Quantitative PCR qpcr - 2 -
The principles of real-time PCR How does a real-time cyler work? 4) Light detector (CCD, PMT) 1) Excitation source (halogen lamp, laser, LEDs) 3) Filters (emission) light light Absorption Emission 2) Filters (excitation) Wavelength - 3 -
The principles of real-time PCR The amplification plot SIGNAL (normalized fluorescence) Real-Time Amplification Plot Initial lag phase no signal detected log phase signal ~doubles every cycle high copy target (e.g. ~10 8 copies) plateau phase reaction slows & stops as a component becomes limiting low copy target (e.g. ~10 3 copies) End point (not quantitative) threshold of detection Low C T = higher copy no. amplifies sooner High C T = lower copy no. amplifies later TIME (amplification cycles) C T = threshold cycle (fractional cycle number where signal crosses a threshold of detection) - 4 -
Design of real-time PCR Cyclers There are two principal technologies to design a real time PCR system Citations from: Real time PCR: Current technology and applications, Logan, Edwards, Saunders (Eds), 2009 1) Block cyclers : Heating/cooling by Peltier elements under the block used e.g. in ABI, Roche LC 480, Eppendorf, Stratagene and Bio-RAD cyclers Peltier Peltier 2) Air cyclers : Each tube spins in a chamber of moving air, keeping all samples at precisely the same temperature during rapid thermal cycling. e.g. used in Roche LC 1.5 & 2.0 and the Rotor-Gene - 5 -
Limitations of block based cyclers Thermal precision (uniformity) in a block-based cycler Typical >0.50 ºC variance across the 96 well block Corner and edge wells most affected Influence of temperature in PCR: Annealing of primers influenced by only small differences in temperature Peltier Peltier Fluorescence 1/Temp Localized hotspots indicating failing Peltier devices The Rotor-Gene Q does not use Peltier devices! Thermal specifications: Uniformity: ±0.01 C, Resolu tion: ±0.02 C - 6 -
Limitations of block based cyclers Optical precision in a block-based cyclers Lamp Shutter Heat Reflecting Mirror Optics in block cyclers general scheme Excitation filter Beam splitter Emission filter Lens CCD Camera Fresnel Lens Well Lenses/heated cover Sample Well Block Multiple optical paths in block systems are complex, error prone and need frequent calibration Passive reference dyes such as ROX are often required to compensate for deviations The Rotor-Gene Q has a single, short optical path and does not need ROX - 7 -
How to overcome technical limitations The rotary principle makes the difference 400 RPM Samples spin continuously during a run 400 RPM (heating or cooling) High-speed data collection all samples read in one revolution (0.15 sec) G-force keeps reagent at base of tube removes bubbles & condensation will not pellet components >10 G Continuous movement means no variation highest well-to-well thermal and optical uniformity - 8 -
Rapid air based cycling Heating Heating mechanism Centrifugal fan drives air around chamber Heater elements switch on Chamber vent seals to contain air Note: holes in the rotor allow free airflow - 9 -
Rapid air based cycling Cooling Cooling mechanism Centrifugal fan drives air around chamber Heater elements switch off Chamber vent opens expelling hot air Centrifugal fan Drives air into chamber Note: holes in the rotor allow free airflow Cool air in - 10 -
Rotary optics Instrument cross-section Reaction Chamber Detection Filters Lens PMT Detector Assembly Tubes in Rotor Spin Past Optics LED Light Source Assembly Spindle/Motor Assembly - 11 -
Quantitation on the Rotor-Gene Q Technicial design features for superior results in qpcr Accurate quantitation Twofold dilutions of human gdna 30 ng (10000 copies) to 0.06 ng (20 copies) TaqMan assay for IL1R2 (no ROX) Accurate CT difference of 1.07 cycles Precise measurements Human gdna; 72 technical replicates TaqMan assay for BCL2 (no ROX) Average CT value was 24.94 ± 0.05 Equivalent to a CV of only 0.2% - 12 -
Reproducibility 96-well block vs. 72-well Rotor Block cycler Rotor-Gene GAPDH gene detected using 6-FAM TaqMan probe Min CT = 18.04, Max CT = 19.51 Cycle difference = 1.50 Std Dev for CT Values = + 0.370 Min CT= 19.02, Max CT = 19.42, Cycle difference = 0.30 Std Dev for CT values = + 0.096-13 -
C T Value Replicates: 96-well block vs. 72-well Rotor Note random spikes & perimeter effect CT Std Dev CT Min Max Cycle range Block + 0.370 18.04 19.51 1.47 Rotor + 0.096 19.02 19.42 0.30-14 -
Optical range & multiplexing Unmatched versatility in dyes and channels Supports all detection systems: Intercalating dyes (e.g. SYBR Green) Hydrolysis probes (TaqMan) Hybridization probes (FRET) Up to 6 individual channels New virtual excitation/detection combinations allow even higher plex Excitation (LEDs) Detection (Filters) - 15 -
Streamline Streamline your workflow Successful PCR needs a good cycler and good chemistry mirna other... Epigenetics Gene Expression Genotyping Pathogen Detection Veterinary Diagnostics Gene expression analysis - various fast-cycling Rotor-Gene kits - all QuantiTect/QuantiFast kits Pathogen detection - CE-IVD certified Rotor-Gene Q artus assays* - QuantiTect Virus kit Veterinary diagnostics - cador assays Genotyping - Type-it Probe PCR kits - Type-it HRM PCR kits Epigenetics - EpiTect MethyLight kits - EpiTect HRM PCR kits mirna research - miscript kits & assays and many more to come... *Note: artus RG (RT)-PCR Kits are not available for diagnostic procedures in USA/Canada - 16 -
Workflow example: Gene expression analysis Automation QIAcube QIAxcel QIAgility Rotor-Gene Q Sample preparation (stabilization, disruption,...) RNA purification & cdna synthesis Assay set-up Real-time RT-PCR analysis Sample & Assay RNAlater RNAprotect Allprotect PAXgene QIAshredder RNeasy Kits RNeasy Plus Kits AllPrep Kits QuantiTect RevT QuantiTect WTA Rotor-Gene Kits QuantiTect Primer Assay Plates QuantiFast Kits QuantiTect Kits - 17 -
An optimized combination! Technical features of Rotor-Gene Kits Optimized NH + 4 buffer system with Q-Bond TM Synthetic Factor MP for multiplexing One procedure for every detection method (SYBR Green, probe, and multiplexing) Reliable one-step and two-step RT-PCR solutions Optimized protocols matching perfectly the Rotor-Gene Q All kits available as 400 rxns standard and 80 rxns trial kits.obtain precision, sensitivity, and reproducibility.by combining QIAGEN s fast qpcr kits with the Rotor-Gene Q - 18 -
Application example: SYBR green based detection in qpcr No optimization and specific amplification Supplier R Nonspecific amplification even after optimization Detection of 5 tenfold dilutions of template Detection of only 4 tenfold dilutions of template Rotor-Gene Q + Rotor-Gene SYBR Green RT-PCR Kit + QuantiTect Primer Assay (BCL2) Cycler and SYBR Green Kit from Supplier R + QuantiTect Primer Assay (BCL2) Good sensitivity and specificity without optimization need for the combination of Rotor-Gene Q and Rotor-Gene SYBR Green RT-PCR Kit - 19 -
Application example: Multiplex qpcr Tenfold dilutions of human leukocyte cdna (100 ng to 10 pg) for 4-plex real-time PCR Supplier S TNF 29,00 TNF 28,00 27,00 IFNG CT-values 26,00 25,00 IFNG 24,00 23,00 No signal detected 22,00 TNFa TNFa IFNG IFNG cmyc2 cmyc2 HSP89 HSP89 Rotor-Gene Q 5Plex + Rotor-Gene Multiplex PCR Kit + TaqMan Assays for TNF, IFNG, MYC, HSP90AA1 QIAGEN Suppplier S Cycler and Multiplex Kit from supplier S + TaqMan Assays for TNF, IFNG, MYC, HSP90AA1 Greater sensitivity and 100% robust performance with the combination Rotor-Gene Q and Rotor-Gene Multiplex PCR Kit - 20 -
mirna research Our complete offering Automation Sample preparation (stabilization, disruption,...) RNA purification & cdna synthesis Assay set-up Real-time RT-PCR analysis Sample & Assay RNAlater RNAprotect Allprotect QIAshredder mirneasy Mini Kit mirneasy FFPE Kit mirneasy Protect Animal Blood Kit mirneasy 96 Kit miscript SYBR Green PCR Kit miscript Primer Assays miscript Primer Assay Plates miscript Primer Assay Sets (Human, Mouse, Rat) - 21 -
RGQ and miscript SYBR Green PCR Kit Perfect combo to study gene regulation Specific quantification over a wide linear range Linear and accurate quantification of mirnas of interest 0.55 0.50 0.45-0.2 10-0.4 10-0.6 10 0.9 0.8 40 35 mir-18 mir-25 Linear (mir-15) mir-15 Linear (mir-18) Linear (mir-25) N o r m. F lu o r o. 0.40 0.35 0.30 0.25 N or m. F lu or o. -0.8 10-1.0 10-1.2 10-1.4 10 d F /dt 0.7 0.6 0.5 Mean C t 30 25 0.20 0.15-1.6 10-1.8 10 0.4 0.3 20 0.10 0.05 Threshold 0.00-2.0 10-2.2 10-2.4 10 Threshold 0.2 0.1 15-2.00-1.00 0.00 1.00-0.05 5 10 15 20 25 30 35 40 Cycle 5 10 15 20 25 30 35 40 Cycle 0.0 60 65 70 75 80 85 90 95 ºC Log [ng] template Rotor-Gene Q + miscript SYBR Green PCR Kit + miscript Control Assay (RNU6B) Template: HeLa S3 cdna (10 ng, 1ng, 0.1 ng, 0.01 ng, 0.001ng) Template: HeLa S3 cdna (10 ng, 1ng, 0.1 ng, 0.01 ng, 0.001ng) Accurate quantification over several logs of template Specific quantification over a wide linear range No need to optimize reaction and cycling conditions - 22 -
Workflow example: Genotyping Automation QIAcube EZ1 advanced QIAsymphony QIAgility Rotor-Gene Q Sample collection & stabilization DNA purification Assay set-up Analysis Sample & Assay QIAcard PAXgene AllProtect QIAamp Kits DNeasy Kits QIAsymphony Blood EZ1 Kits Type-it Fast SNP PCR Kit Type-it HRM PCR Kit - 23 -
Application example: Genotyping with dual labeled probes Allelic discrimination plot analysis with a panel of 70 different genomic DNAs (1 ng each in 10 μl reactions) Rotor-Gene Q + Type-it Fast SNP Probe PCR Kit + TaqMan SNP genotyping assay for rs 951134 Tight clustering and reliable genotyping results even with low amounts of template using probe based genotyping and the Rotor-Gene Q - 24 -
Application example: Genotyping with HRM Accurate SNP genotyping by HRM 105 100 95 90 85 80 75 70 Homozygote mutant 15 Homozygote mutant 10 Normalised Fluorescence 65 60 55 50 45 Heterozygote Normalised minus wt 5 Heterozygote 40 35 30 25 20 15 10 Wild Type 0 Wild Type 5 78,0 78,5 79,0 79,5 80,0 80,5 81,0 81,5 82,0 82,5 83,0 83,5 84,0 84,5 85,0 85,5 86,0 86,5 87,0 deg. Standard normalized melt curve Rotor-Gene HRM 5plex + Type-it HRM PCR Kit + 10 ng genomic DNA 78,0 78,5 79,0 79,5 80,0 80,5 81,0 81,5 82,0 82,5 83,0 83,5 84,0 84,5 85,0 85,5 86,0 86,5 87,0 deg. Difference plot normalized to a wild type sample Genotyping with HRM on the Rotor-Gene Q is an emerging fast and cost-effective technique with high specificity - 25 -
Into to HRM General concept What is High Resolution Melt analysis? HRM = closed-tube, post-pcr analysis, that characterizes double stranded PCR products based on their dissociation (melting) behaviors. Distinguishes samples with different sequence, length, GC content Sample characterization by melt curve shape and melt temperature Similar to classical melting curve analysis, but provides far more information Fluorescence Temperature HRM monitors the loss of fluorescence of saturating intercalating dyes (EvaGreen, LCGreen) during DNA dissociation - 26 -
Into to HRM HRM Chemistry Intercalating dyes provide a proportion decrease in fluorescence as the strands begin to dissociate. New intercalating dyes such as LCGreen EvaGreen and Syto9 are less toxic than SYBR. High concentrations for DNA saturation eliminates potential for dye relocation and is ideal for HRM - 27 -
Introduction to HRM Sample characterization by melt curve shape and melt temperature Allele 1 Allele 2 Allele 1 Allele 2 Mutant T A T A weaker binding lower melting point C A T G Heterozygote C G C G stronger binding higher melting point Hybrids with very weak binding yielding lowest melting point Wild Type T A C G mixture with intermediate melting point Homozygotes are easily differentiated by different Tm Heterozygotes form various heteroduplexes resulting in a different melt curve shape - 28 -
Introduction to HRM What is necessary for high performance HRM? HRM instruments collect fluorescent signals with much greater optical and thermal precision than previous methods and require: High intensity illumination ideally with a specially tuned HRM channel High sensitivity optical detection Fast data acquisition rate Absolute minimum of sample-to-sample thermal and optical variation A dedicated software module for the analysis The Rotor-Gene Q (as an air-cycler) meets these demands for accurate HRM! Conventional (block-based) real-time systems are not optimal for HRM 1) Block cyclers : 2) Air cyclers : Peltier Peltier - 29 -
Introduction to HRM How good can it be? Benchmarking by SNP classes SNP Class Base Change Typical Tm Shift Rarity (in humans) I C/T and G/A Large>0.5 C 64% II C/A and G/T 20% III IV C/G A/T Very Small <0.2 C 9% 7% SNP classes Venter et al 2002 Example of a class IV SNP on the Rotor Gene Herrmann et al Clinical Chemistry 53, 2007-30 -
Instrument comparison HRM data Herrmann et al 2007 Clinical Chemistry 53, http://www.clinchem.org/cgi/content/abstract/clinchem.2007.088120v1 Expanded Instrument Comparison of Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping Fig 1 (detail) Normalized melting curves of a 110 bp β-globin amplicon (triplicate HRM data) 100 Normalized Fluorescence (%) 50 homozygous wild type homozygous mutant (c.20a>t) single heterozygous mutant (c.20a>t) double heterozygous mutant [9C>T; 20A>T] LightScanner (384-well) 0 80 85 90 Temperature ( C) - 31 -
Instrument comparison HRM data Herrmann et al 2007 Clinical Chemistry 53, http://www.clinchem.org/cgi/content/abstract/clinchem.2007.088120v1 Expanded Instrument Comparison of Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping Fig 1 (detail) Normalized melting curves of a 110 bp β-globin amplicon (triplicate HRM data) Normalized Fluorescence (%) 100 50 0 80 85 90 Temperature ( C) homozygous wild type homozygous mutant (c.20a>t) single heterozygous mutant (c.20a>t) double heterozygous mutant [9C>T; 20A>T] LightCycler 480 (384-well) - 32 -
Instrument comparison HRM data Herrmann et al 2007 Clinical Chemistry 53, http://www.clinchem.org/cgi/content/abstract/clinchem.2007.088120v1 Expanded Instrument Comparison of Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping Fig 1 (detail) Normalized melting curves of a 110 bp β-globin amplicon (triplicate HRM data) Normalized Fluorescence (%) 100 50 homozygous wild type homozygous mutant (c.20a>t) single heterozygous mutant (c.20a>t) double heterozygous mutant [9C>T; 20A>T] 7300 Real-Time PCR System (96-well) 0 80 85 90 Temperature ( C) - 33 -
Instrument comparison HRM data Herrmann et al 2007 Clinical Chemistry 53, http://www.clinchem.org/cgi/content/abstract/clinchem.2007.088120v1 Expanded Instrument Comparison of Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping Fig 1 (detail) Normalized melting curves of a 110 bp β-globin amplicon (triplicate HRM data) Normalized Fluorescence (%) 100 50 homozygous wild type homozygous mutant (c.20a>t) single heterozygous mutant (c.20a>t) double heterozygous mutant [9C>T; 20A>T] Mastercycler ep realplex 4S (96-well) 0 80 85 90 Temperature ( C) - 34 -
Instrument comparison HRM data Herrmann et al 2007 Clinical Chemistry 53, http://www.clinchem.org/cgi/content/abstract/clinchem.2007.088120v1 Expanded Instrument Comparison of Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping Fig 1 (detail) Normalized melting curves of a 110 bp β-globin amplicon (triplicate HRM data) Normalized Fluorescence (%) 100 50 0 80 homozygous wild type homozygous mutant (c.20a>t) single heterozygous mutant (c.20a>t) double heterozygous mutant [9C>T; 20A>T] 85 90 Temperature ( C) Rotor-Gene Real-Time Analyzer - 35 -
Intro to HRM Main applications of HRM SNP genotyping Simple, fast, cost-effective point mutation detection by amplifying the SNP region followed by an accurate melt Epigenetics: Quantitative methylation analysis Bisulfite conversion translates methylation status in sequence variance, which can be detected quantitatively by HRM Gene or mutation scanning Easily find heteroduplexes from heterezygotes by the distortion of the melting curve Sequence matching If samples are not identical different heteroduplexes are formed that change the shape of the melting curve (HLA, forensics) - 36 -
Application example: Epigenetic research with HRM Various ratios of methylated and unmethylated DNA-APC 100 95 90 35 30 100% methylated 85 Normalised Fluorescence 80 75 70 65 60 55 50 45 40 35 30 25 20 15 100% methylated 90%. 70% 50%. 30%. 10%. Normalised minus 50%m50%u 25 20 15 10 5 0-5 -10-15 -20 90%. 70% 50%. 30%. 10%. 10 5 0% methylated 0 75,0 75,5 76,0 76,5 77,0 77,5 78,0 78,5 79,0 79,5 80,0 80,5 81,0 81,5 82,0 82,5 83,0 83,5 84,0 deg. -25 0% methylated 75,0 75,5 76,0 76,5 77,0 77,5 78,0 78,5 79,0 79,5 80,0 80,5 81,0 81,5 82,0 82,5 83,0 83,5 84,0 deg. Standard normalized melt curve Rotor-Gene HRM + EpiTect HRM PCR Kit Difference plot normalized to a 50% methylated sample Excellent performance for quantitative methylation screening (fast & cost effective) - 37 -
Application example: Epigenetic research with HRM from T. Wojdacz et al, Nucl. Acid Res. 2007 35(1):e41 12 CpG s/ 109bp Determination of the methylation status of the MGMT promoter Low level methylation 0.1% sensitivity 5CpG s/ 94bp - 38 -
HRM Application Example Mutation Screening from M.Krypuy et al, BMC Cancer. 2006 Dec 21;6(1):295 KRAS codon 12 and 13 mutations in non-small cell lung cancer - 39 -
Main benefits of HRM Advantages of HRM Very cost effective versus other genotyping technologies such as sequencing and probe based SNP-typing Fast and sensitive: huge number of samples in rapid time Simple and convenient: can be performed in any lab with access to a HRM analyzer No transfer of samples: rapid cycle PCR followed by HRM analysis Sample is not consumed, you can do sequencing afterwards Unlimited application range - 40 -
Rotor-Gene Q Pure Detection Highly versatile real-time PCR and High Resolution Melting system Outstanding thermal and optical performance due to rotary format Unmatched optical range from UV to infrared for up to 6 channels State-of-the art analyses supported by user-friendly software Low maintenance and maximum convenience due to robust design High performance in multiple applications with QIAGEN kits & assays - 41 -
Question time - 42 -