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qPCR SNAPSHOTS LIVE. MAY 13, 2003. Agenda. Introduction Objectives & Methodologies Detection Chemistries Instrumentation Conclusion. Real-Time PCR. Ability to detect PCR product as it is synthesized
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qPCR SNAPSHOTS LIVE MAY 13, 2003
Agenda • Introduction • Objectives & Methodologies • Detection Chemistries • Instrumentation • Conclusion
Real-Time PCR • Ability to detect PCR product as it is synthesized • Requires fluorescence-based detection chemistries and specialized detection instrumentation • Advantages: • Increased analytical sensitivity • Faster results • Broad applicability Introduction
Study Demographics 44% Academic 21% Pharmaceutical/Biotechnology Hospital or University Medical Center 18% Government 8% 5% Private Research 2% Contract Research 2% Commercial Testing Lab <1% Medical Device/Diagnostics <1% Healthcare Network/Facility 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% Introduction
Quantitative vs. Qualitative Applications Qualitative only Both (qualitative and 11% quantitative) 58% Quantitative only 31% Objectives & Methodologies
Template Use Only RNA or cDNA as a Only DNA as a template template 17% 49% DNA, cDNA and RNA as templates 34% Objectives & Methodologies
Research Objectives 24% Gene expression- primary validation 14% 17% Gene expression-confirmation of microarray data 13% 13% Viral load detection 10% 10% Bacterial detection/ identification 14% 11% Gene duplication or other DNA quantification 10% 9% Allelic discrimination 13% 5% Other 11% DNA, cDNA and RNA as a template Only DNA as a template 5% SNP genotyping 4% 0% 5% 10% 15% 20% 25% 30% Objectives & Methodologies
Kits vs. Components: Preferences by Template Both RNA 13% A commercially available qRT-PCR kit 53% Individual components 34% Individual components 21% A commercially available DNA qPCR kit (or qPCR master mix) 57% Both 22% Objectives & Methodologies
Convenience Guaranteed optimized system Cost effective Greater flexibility Less expensive Have own system Too little reagent volume in kits Kits vs. Components: Reasons for Selection Kits Components Objectives & Methodologies
Detection Chemistry Preferences Fluorescent probes or primers only SYBR Green only 36% 31% Both SYBR Green and fluorescent probes or primers 33% Detection Chemistries
Commonly Used Fluorescent Probes 78% TaqMan probes 19% Molecular Beacons 15% FRET probes LUX fluorogenic primers 9% 9% MGB Eclipse probes 3% Other 2% Scorpion probes 0% 10% 20% 30% 40% 50% 60% 70% 80% Detection Chemistries
Sensitivity Linear dynamic range Time-to-results Throughput Software Block/sample capacity Size User interface Factors Influencing Instrumentation Purchase Performance Parameters Product Specifications
Commonly Used Real-Time PCR Instrumentation 26% ABI PRISM 7700 Sequence Detection System 18% ABI PRISM 7000 Sequence Detection System 16% LightCycler System iCycler iQ Real Time PCR Detection System 12% ABI PRISM 7900HT Sequence Detection System 9% 7% GeneAmp 5700 Sequence Detection System 3% Smart Cycler System 2% DNA Engine Opticon (or Opticon 2) System 2% Rotor-Gene 2% Mx4000 Multiplex Quantitative PCR System 1% Other <1% R.A.P.I.D. System 0% 5% 10% 15% 20% 25% 30% Instrumentation
Conclusions • Real-time PCR is dominated by gene expression studies and quantitative applications. • The majority of researchers prefer using kits rather than individual components for real-time PCR amplification. • Fluorescent probes/primers and SYBR Green dye are nearly equal in popularity as detection chemistries. • 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.
