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Quantitative real-time RT-PCR – a perspective

S A Bustin

S A Bustin Institute of Cellular and Molecular Science, Barts and the London, Queen Mary’s School of Medicine and Dentistry, University of London, London, UK
EMBL Heidelberg, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
Stratagene Europe, PO Box 12085, 1100 AB Amsterdam, The Netherlands
Abteilung Physiologie, Zentralinstitut für Ernährungs und Lebensmittelforschung, Technische Universität München, Weihenstephaner Berg 3, D-85354 Freising-Weihenstephan, Germany

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V Benes

V Benes Institute of Cellular and Molecular Science, Barts and the London, Queen Mary’s School of Medicine and Dentistry, University of London, London, UK
EMBL Heidelberg, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
Stratagene Europe, PO Box 12085, 1100 AB Amsterdam, The Netherlands
Abteilung Physiologie, Zentralinstitut für Ernährungs und Lebensmittelforschung, Technische Universität München, Weihenstephaner Berg 3, D-85354 Freising-Weihenstephan, Germany

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T Nolan

T Nolan Institute of Cellular and Molecular Science, Barts and the London, Queen Mary’s School of Medicine and Dentistry, University of London, London, UK
EMBL Heidelberg, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
Stratagene Europe, PO Box 12085, 1100 AB Amsterdam, The Netherlands
Abteilung Physiologie, Zentralinstitut für Ernährungs und Lebensmittelforschung, Technische Universität München, Weihenstephaner Berg 3, D-85354 Freising-Weihenstephan, Germany

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M W Pfaffl

M W Pfaffl Institute of Cellular and Molecular Science, Barts and the London, Queen Mary’s School of Medicine and Dentistry, University of London, London, UK
EMBL Heidelberg, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
Stratagene Europe, PO Box 12085, 1100 AB Amsterdam, The Netherlands
Abteilung Physiologie, Zentralinstitut für Ernährungs und Lebensmittelforschung, Technische Universität München, Weihenstephaner Berg 3, D-85354 Freising-Weihenstephan, Germany

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The real-time reverse transcription polymerase chain reaction (RT-PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction. This combines the nucleic acid amplification and detection steps into one homogeneous assay and obviates the need for gel electrophoresis to detect amplification products. Use of appropriate chemistries and data analysis eliminates the need for Southern blotting or DNA sequencing for amplicon identification. Its simplicity, specificity and sensitivity, together with its potential for high throughput and the ongoing introduction of new chemistries, more reliable instrumentation and improved protocols, has made real-time RT-PCR the benchmark technology for the detection and/or comparison of RNA levels.