A new mathematical model for relative quantification in real-time RT-PCR.
Journal: 2002/November - Nucleic Acids Research
ISSN: 1362-4962
PUBMED: 11328886
Abstract:
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
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Nucleic Acids Res 29(9): e45

A new mathematical model for relative quantification in real-time RT–PCR

Institute of Physiology, FML-Weihenstephan, Center of Life and Food Sciences, Technical University of Munich, Germany
To whom correspondence should be addressed at present address: Institut für Physiologie, Weihenstephaner Berg 3, 85354 Freising, Weihenstephan, Germany. Tel: +49 8161 71 3511; Fax: +49 8161 71 4204; Email: ed.nahpetsnehiew@lffafp
Received 2000 Dec 18; Revised 2001 Feb 21; Accepted 2001 Mar 14.

Abstract

Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.

Abstract

Determination of variation was done in 20 ng reverse transcribed total RNA. Test variation is based on CP variation and expressed as mean CP with CV.

ACKNOWLEDGEMENTS

The author thanks D.Schmidt for technical assistance. Primers, primer sequences and samples were kindly donated by Drs S.Wegener and W.Mann in collaboration with the BioChip division of MWG Biotech in Ebersberg, Germany.

ACKNOWLEDGEMENTS

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