Development and Validation of an Externally Standardised Quantitative Insulin-like Growth Factor-1 RT-PCR Using LightCycler SYBR Green I Technology
- Michael PfafflAffiliated withInstitute of Physiology, Research Centre for Milk & Food, Technical University of Munich
Internally standardised competitive RT-PCR measured by HPLC separation and UV detection  or high resolution gel electrophoresis followed by densitometric analysis : In a competitive RT-PCR, a reference RNA mutant is reverse transcribed and co-amplified in the same reaction tube with the native mRNA sequence of interest. Internally standardised RT-PCR is a very time-consuming and laborious technique. It is generally believed to yield the most precise results, because all parameters throughout RT-PCR act on both the analyte and reference mutant.
Externally standardised RT-PCR with online-detection using LightCycler SYBR Green I technology [6, 13]: LightCycler PCR with SYBR Green I online detection produces reliable and rapid results. Because it uses an external standard curve, the amplification efficiencies for the calibration curve and the analyte must be equal for accurate quantification.
Externally standardised RT-PCR with online-detection using specific LightCycler hybridisation probes : This detection format is based on fluorescence resonance energy transfer.
- Development and Validation of an Externally Standardised Quantitative Insulin-like Growth Factor-1 RT-PCR Using LightCycler SYBR Green I Technology
- Book Title
- Rapid Cycle Real-Time PCR
- Book Subtitle
- Methods and Applications
- Book Part
- pp 281-291
- Print ISBN
- Online ISBN
- Springer Berlin Heidelberg
- Copyright Holder
- Springer-Verlag Berlin Heidelberg
- Additional Links
- Industry Sectors
- eBook Packages
- Editor Affiliations
- 1. Institut für Immunologie, Ruprecht Karls-Universität Heidelberg
- 2. Department of Pathology, University of Utah Medical School
- 3. Sendai, Japan
- Michael Pfaffl (4) (5)
- Author Affiliations
- 4. Weihenstephaner Berg 5, 85354, Freising-Weihenstephan, Germany
- 5. Institute of Physiology, Research Centre for Milk & Food, Technical University of Munich, Germany
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